Multi-dye compounds

Abstract

Multiplex dye compounds and methods of use thereof are described herein, inter alia.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 356,863, filed on June 29, 2022; U.S. Provisional Application No. 63 / 356,874, filed on June 29, 2022; U.S. Provisional Application No. 63 / 408,665, filed on September 21, 2022; and U.S. Provisional Application No. 63 / 453,546, filed on March 21, 2023; each of which is hereby incorporated by reference in its entirety and for all purposes. Background Art

[0003] Current qPCR probe designs have a reporter dye at the 5' end and a quencher molecule at the 3' end. There are typically 20-30 bases between the fluorophore and the quencher. An internal quencher can be placed within 6-12 bases between the reporter dye and the quencher, closer to the fluorophore dye. This shortened length provides better quenching and lower background, which in turn improves the accuracy and robustness of the assay. Disclosed herein are solutions to these and other problems in the art. Summary of the Invention

[0004] In one aspect, compounds or salts thereof having the following formula are provided:

[0005]

[0006] B is a divalent nucleobase.

[0007] L 1 It is a divalent linker.

[0008] R 2 Is it hydrogen or -OR 2A .

[0009] R 3 Yes - OR 3A or -OP(NR 3B R 3C )-OR 3A .

[0010] R 4 is hydrogen or unsubstituted methyl, or R 2 and R 4 Substituents connect to form substituted or unsubstituted heterocyclic alkyl groups.

[0011] R 5 Yes-OR 5A .

[0012] R 1 and R 10are independently hydrogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl.

[0013] R 6 、R 7 、R 8 and R 9 are independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3R A -SO2NH2, -NHNH2, -ONH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCBr3, -OCF3, -OCI3, -OCH2Cl, -OCH2Br, -OCH2F, -OCH2I, -OCHCl2, -OCHBr2, -OCHF2, -OCHI2, -SF5, -N3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0014] R 1 and R 6 They can be linked to form substituted or unsubstituted heterocyclic alkyl groups, or substituted or unsubstituted heteroaryl groups.

[0015] R 8 and R 10 They may be linked to form a substituted or unsubstituted heterocycloalkyl group, or a substituted or unsubstituted heteroaryl group.

[0016] R 2A 、R 3A 、R 3B 、R 3C 、R 5A and R Aand -CHCl, -CBr, -CF, -CI, -CHCl, -CHBr, -CHF, -CHI, -CHCl, -CHBr, -CHF, -CHI, -CN, -OH, -NH, -COOH, -CONH, -OCCl, -OCF, -OCBr, -OCI, -OCHCl, -OCHBr, -OCHI, -OCHF, -OCHCl, -OCHBr, -OCHI, -OCHF, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0017] In one aspect, an oligonucleotide having the formula:

[0018] B.L 1 、R 1 、R 2 、R 4 、R 6 、R 7 、R 8 、R 9 and R 10 As described herein, it is included in the implementation scheme.

[0019] L 5 It is a bivalent oligonucleotide linker containing 4 to 40 nucleotides.

[0020] L 50 It is a bond, -NH-, -O-, -S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene.

[0021] R 50 is the detectable portion.

[0022] R 30 Yes - OR 30A or -OP(NR 3B R 3C )-OR 3A .

[0023] R 30A is hydrogen, a 3' blocking moiety, or a monovalent oligonucleotide moiety.

[0024] R3A 、R 3B and R 3C Independently, it is hydrogen, -CCl3, -CBt3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0025] In one aspect, a method of forming a double-stranded nucleic acid is provided, the method comprising contacting a target oligonucleotide with a nucleic acid probe comprising a quencher moiety (e.g., an internal quencher moiety), thereby forming a double-stranded nucleic acid; wherein the nucleic acid probe has the formula:

[0026] (XIX) or a salt thereof. B, L 1 , L 5 , L 50 、R 2 、R 4 and R 50 As described herein, included in the embodiments. 30 Yes - OR 30A , where R 30A Q is the monovalent oligonucleotide moiety. Q is the quencher moiety (e.g., the internal quencher moiety).

[0027] In an embodiment, Q is a quencher moiety having the formula:

[0028] R 1 、R 6 、R 7 、R 8 、R 9 and R 10 As described herein, it is included in the implementation scheme.

[0029] In one aspect, a method for detecting nucleic acids in a sample is provided, the method comprising: (i) providing a reaction mixture comprising: at least a portion of the sample; a first probe detectably labeled with a first label, configured to generate a first fluorescent signal; and a second probe detectably labeled with a second label, configured to generate a second fluorescent signal, wherein the first and second probes have different sequences, wherein the first and second labels are identical and / or generate substantially the same fluorescence; and wherein the second probe has the following formula:

[0030] (XIX) or a salt thereof, wherein Q, B, L 1 、L 5 、L 50 、R 2 and R 4 As described herein, it is included in the implementation scheme; R 30 Yes-OR 30A , where R 30A is the monovalent oligonucleotide portion; R 50 (ii) subjecting the reaction mixture to an amplification process comprising a first set of reaction conditions and a second set of reaction conditions, the first set of reaction conditions being different from the second set of reaction conditions; and (iii) determining the presence of a first and / or second nucleic acid target in the sample by measuring fluorescence signals during the first set of reaction conditions, the fluorescence signals during the first set of reaction conditions being correlated with a specific interaction or lack thereof between the first probe and the first nucleic acid target, measuring fluorescence signals during the second set of reaction conditions, the fluorescence signals during the second set of reaction conditions being correlated with a specific interaction or lack thereof between the first probe and the first nucleic acid target, and estimating the presence and / or amount of the first and second nucleic acid targets.

[0031] In one aspect, a method for detecting nucleic acids in a sample is provided, the method comprising: (i) providing a reaction mixture comprising: at least a portion of the sample; a first probe detectably labeled with a first label, configured to generate a first fluorescent signal indicating the presence or absence of a first nucleic acid target; and a second probe detectably labeled with a second label, configured to generate a second fluorescent signal indicating the presence or absence of a second nucleic acid target, wherein the first and second probes have different sequences, wherein the first and second labels are identical and / or generate substantially the same fluorescence; and wherein the second probe has the following formula:

[0032] (XIX) or its salts, wherein Q, B, L 1 、L 5 、L 50 、R 2 and R 4As described herein, it is included in the implementation scheme; R 30 Yes-OR 30A , where R 30A is the monovalent oligonucleotide portion; R 50 is a second label; (ii) subjecting the reaction mixture to an amplification process comprising a first set of reaction conditions and a second set of reaction conditions, the first set of reaction conditions being different from the second set of reaction conditions; and (iii) determining the presence and / or amount of the first and / or second nucleic acid target in the reaction mixture by measuring a first total fluorescent signal including any first fluorescent signal (if any) during the first set of reaction conditions, measuring a second total fluorescent signal including any first fluorescent signal (if any) and any second fluorescent signal (if any) during the second set of reaction conditions, and estimating the first fluorescent signal and / or the second fluorescent signal based on the first total fluorescent signal and the second total fluorescent signal.

[0033] In one aspect, a method for detecting nucleic acid in a sample is provided, the method comprising: providing a reaction mixture comprising a primer pair complementary to a nucleic acid target or its complement for producing an amplicon and a non-cleavable probe configured to hybridize to the amplicon, the probe comprising a detectable label configured to provide a fluorescent signal corresponding to the amount of amplicon produced, subjecting the reaction mixture to an amplification process to produce the amplicon, wherein the label produces fluorescence and the probe is not cleaved during the amplification process, and wherein the amplification process utilizes a series of thermal cycling steps comprising at least three different target temperatures; and measuring the fluorescent signal from the probe; wherein the probe has the formula:

[0034] (XIX) or its salts, wherein Q, B, L 1 、L 5 、L 50 、R 2 and R 4 As described herein, it is included in the implementation scheme; R 30 Yes-OR 30A , where R 30A is the monovalent oligonucleotide portion; R 50 is a detectable label.

[0035] In one aspect, a method for detecting nucleic acid in a sample is provided, the method comprising: providing a reaction mixture comprising a primer pair targeting a nucleic acid target for producing an amplicon, the primer pair comprising a tailed primer and a tailless primer provided at different concentrations, and a non-cleavable probe configured to hybridize with the amplicon, the probe comprising a detectable label configured to produce a fluorescent signal corresponding to the amount of the amplicon produced, subjecting the reaction mixture to an amplification process to produce the amplicon, wherein the probe produces fluorescence without being cleaved during the amplification process; and measuring the fluorescent signal from the probe; wherein the probe has the formula:

[0036] (XIX) or its salts, wherein Q, B, L 1 、L 5 、L 50 、R 2 and R 4 As described herein, it is included in the implementation scheme; R 30 Yes-OR 30A , where R 30A is the monovalent oligonucleotide portion; R 50 is a detectable label.

[0037] In one aspect, a method for detecting the presence or amount of a first and / or second target in a reaction mixture is provided, the method comprising: comprising a first probe and a second probe, wherein the first probe can specifically interact with the first target and comprises a first label that can produce a first detectable signal, and the second probe can specifically interact with the second target and comprises a second label that can produce a second detectable signal; wherein the second probe has the following formula:

[0038] (XIX) or its salts, wherein Q, B, L 1 、L 5 、L 50 、R 2 and R 4 As described herein, it is included in the implementation scheme; R 30 Yes-OR 30A , where R 30A is a monovalent oligonucleotide moiety; and R 50is a second label; allowing the first probe and the second probe to specifically interact with any first target and second target in the reaction mixture, respectively; measuring a first total signal through a filter under a first set of conditions, wherein the first total signal includes the first detectable signal and the second detectable signal from the first label and the second label, and wherein under the first set of conditions, the first detectable signal is increased due to the specific interaction of the first probe with the first target, but the second detectable signal is not increased due to the specific interaction of the second probe with the second target; measuring a second total signal through the same filter under a second set of conditions, wherein the second total signal includes the first detectable signal and the second detectable signal from the first label and the second label, and wherein under the second set of conditions, the second detectable signal is increased due to the specific interaction of the second probe with the second target; and assessing the presence or amount of the first target and / or the second target by estimating the first detectable signal and the second detectable signal based on both the first total signal and the second total signal. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1A-1B . Figure 1A Shown are the emission spectra of various fluorescent dyes commonly used in nucleic acid detection assays and their associated detection channels. Figure 1B The emission spectra of various fluorescent dyes and their associated detection channels commonly used for nucleic acid detection assays are shown, demonstrating that "Dye 1" and "Dye 2" can be detected in the same channel.

[0040] Figure 2A-2B . Figure 2A is a schematic overview of a method for detecting multiple target nucleic acids within the same detection channel by providing different first and second probe types, varying reaction mixture conditions, and measuring the resulting total signal under each set of conditions. Figure 2B is a graph showing that when the reaction mixture cycles between a first set of reaction conditions and a second set of reaction conditions, Figure 2A A plot of the signal response over time for the method outlined in .

[0041] Figures 3A-3B . Figure 3A The fluorescent activity of cleavable probes (e.g., TaqMan probes) and non-cleavable probes (e.g., extendable fluorescent (EF) probes) during the annealing, extension, and denaturation steps of thermal cycling is shown, showing that the cleavable probes increase the fluorescent signal by releasing and not quenching the corresponding label, while the non-cleavable probes increase the fluorescent signal when duplex amplicons are formed during extension. Figure 3B is a graph showing the fluorescence signal response over time during thermal cycling of an amplification process utilizing Figure 3AThe cleavable and non-cleavable probes can be used to achieve intra-channel multiplexing;

[0042] Figures 4A-4C . Figure 4A The process of using tailed primers specific to nucleic acid targets to form a template that the EF probe can hybridize with is shown. Figure 4B It is shown that the reaction mixture can be included to implement Figure 4A An example of a process is tailing a forward primer, a reverse primer, and an EF probe. Figure 4C Shown is a three-stage thermal cycling method that can be used during an amplification process involving an EF probe and, optionally, a TaqMan probe.

[0043] Figure 5A-5B An overview of methods for detecting multiple target nucleic acids within the same detection channel in digital polymerase chain reaction (dPCR) applications is provided.

[0044] Figures 6A-6F . Figure 6A Fluorescence signals of the TaqMan probe and the extendable fluorescent (EF) probe at the extension and denaturation steps are shown. Figures 6B-6D The results of the double-strand determination test are shown, in which the TaqMan probe and the EF probe were designed to be in the same dye channel ( Figure 6B ) or different dye channels ( Figures 6C-6D ) to generate a fluorescent signal. Figure 6E Will use Figure 6B The baseline-adjusted EF-related fluorescence signal (dRn) obtained from the measurement results is compared with that in Figure 6C The results showed a close correlation between the directly measured EF-related fluorescence signals in the assay, and thus proved that fluorescence signals of different probe types attributable to the same detection channel could be distinguished individually. Figure 6F The baseline-adjusted TaqMan-related fluorescence signals (dRn) were compared.

[0045] Figure 7 The results of another assay including five different detection channels / dyes, each with a corresponding TaqMan probe and EF probe, are shown. The results show that the fluorescence signals of different probe types can be distinguished individually, and that the nine-fold reaction can be carried out effectively.

[0046] Figure 8 This is a graph comparing the endpoint signals of partitions at 65°C and 95°C after the dPCR process, showing that the partition signals fall into identifiable clusters that allow for the estimation of different target concentrations.

[0047] Figure 9Chemical structures of QSY7-dU-Amidite (top panel) and QSY21-dU-Amidite (bottom panel).

[0048] Figure 10 . Sample list of fluorophores and compatible quenchers (shaded box = compatible). DETAILED DESCRIPTION

[0049] I. Definition

[0050] Abbreviations used herein have their conventional meanings in chemistry and biology.Chemical structures and formulae described herein are constructed according to standard rules of chemical valence known in the chemical arts.

[0051] When substituents are designated by their conventional chemical formula as written left to right, they equally encompass the chemically identical substituents that would result from writing the structure right to left, for example, -CH2O- is equivalent to -OCH2-.

[0052] Unless otherwise indicated, the term "alkyl" by itself or as part of another substituent refers to a straight (i.e., unbranched) or branched carbon chain (or carbons), or combinations thereof, which may be fully saturated, monounsaturated, or polyunsaturated, and may include monovalent, divalent, and polyvalent groups. An alkyl group may include a specified number of carbons (e.g., C1-C 10 (This refers to one to ten carbon atoms). In an embodiment, the alkyl group is fully saturated. In an embodiment, the alkyl group is monounsaturated. In an embodiment, the alkyl group is polyunsaturated. The alkyl group is a noncyclic chain. Examples of saturated hydrocarbon groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, methyl, their homologues and isomers, such as n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. Unsaturated alkyl groups are alkyl groups having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propynyl, crotonyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-propynyl and 3-propynyl, 3-butynyl, and higher homologues and isomers. The alkyl group is an alkyl group connected to the rest of the molecule by an oxygen linker (-O-). The alkyl moiety can be an alkenyl moiety. The alkyl moiety may be an alkynyl moiety. Alkenyl groups include one or more double bonds. Alkynyl groups include one or more triple bonds.

[0053] Unless otherwise stated, the term "alkylene" by itself or as part of another substituent refers to a divalent group derived from an alkyl group, such as, but not limited to, -CH2CH2CH2CH2-. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those having 10 or fewer carbon atoms preferred herein. "Lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, typically having eight or fewer carbon atoms. Unless otherwise stated, the term "alkenylene" by itself or as part of another substituent refers to a divalent group derived from an alkene group. Unless otherwise stated, the term "alkynylene" by itself or as part of another substituent refers to a divalent group derived from an alkyne group. In an embodiment, an alkylene group is fully saturated. In an embodiment, an alkylene group is monounsaturated. In an embodiment, an alkylene group is polyunsaturated. An alkenylene group includes one or more double bonds. An alkynylene group includes one or more triple bonds.

[0054] Unless otherwise stated, the term "heteroalkyl" on its own or in combination with another term means a stable straight or branched chain or combination thereof, comprising at least one carbon atom and at least one heteroatom (e.g., O, N, P, Si, and S), wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom (e.g., N, S, Si, or P) may be located at any internal position of the heteroalkyl group or at a position where the alkyl group is attached to the remainder of the molecule. Heteroalkyl groups are acyclic chains. Examples include, but are not limited to: -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -S-CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, -O-CH2-CH3, and -CN. At most two or three heteroatoms can be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. The heteroalkyl moiety may include one heteroatom (e.g., O, N, S, Si, or P). The heteroalkyl moiety may include two optionally different heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may include three optionally different heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may include four optionally different heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may include five optionally different heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may include up to eight optionally different heteroatoms (e.g., O, N, S, Si, or P). Unless otherwise stated, the term "heteroalkenyl" itself, or in combination with another term, means a heteroalkyl group comprising at least one double bond. In addition to one or more double bonds, a heteroalkenyl group may optionally include more than one double bond and / or one or more triple bonds. Unless otherwise stated, the term "heteroyynyl" itself, or in combination with another term, means a heteroalkyl group comprising at least one triple bond. In addition to one or more triple bonds, a heteroyynyl group may optionally include more than one triple bond and / or one or more double bonds. In embodiments, the heteroalkyl group is fully saturated. In embodiments, the heteroalkyl group is monounsaturated. In embodiments, the heteroalkyl group is polyunsaturated.

[0055] Similarly, unless otherwise indicated, the term "heteroalkylene" by itself or as part of another substituent means a divalent radical derived from heteroalkyl, such as, but not limited to, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene, heteroatoms may also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)2R'- represents both -C(O)2R'- and -R'C(O)2-. As described above, heteroalkyl groups as used herein include those groups that are attached to the remainder of the molecule through a heteroatom, such as -C(O)R', -C(O)NR', ​​-NR'R", -OR', -SR' and / or -S02R'. When "heteroalkyl" is recited followed by a specific heteroalkyl group, such as -NR'R", etc., it is understood that the terms heteroalkyl and -NR'R" are not redundant or mutually exclusive. Rather, the specific heteroalkyl group is recited for added clarity. Thus, the term "heteroalkyl" should not be construed herein to exclude specific heteroalkyl groups, such as -NR'R", etc. The term "heteroalkenylene" by itself or as part of another substituent, unless otherwise stated, means a divalent radical derived from heteroalkenylene. The term "heteroalkynyl" by itself or as part of another substituent, unless otherwise stated, means a divalent radical derived from heteroalkynylene. In an embodiment, heteroalkylene is fully saturated. In an embodiment, heteroalkylene is monounsaturated. In an embodiment, heteroalkylene is polyunsaturated. Heteroalkenylene includes one or more double bonds. The pyridyl group includes one or more triple bonds.

[0056] Unless otherwise stated, the terms "cycloalkyl" and "heterocycloalkyl," by themselves or in combination with other terms, mean cyclic versions of "alkyl" and "heteroalkyl," respectively. Cycloalkyl and heterocycloalkyl are not aromatic. Additionally, for heterocycloalkyl, a heteroatom may occupy the position at which the heterocycle is attached to the rest of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridinyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. "Cycloalkylene" and "heterocycloalkylene," by themselves or as part of another substituent, refer to divalent radicals derived from cycloalkyl and heterocycloalkyl, respectively. In embodiments, cycloalkyl is fully saturated. In embodiments, cycloalkyl is monounsaturated. In embodiments, cycloalkyl is polyunsaturated. In embodiments, heterocycloalkyl is fully saturated. In embodiments, heterocycloalkyl is monounsaturated. In embodiments, heterocycloalkyl is polyunsaturated.

[0057] In embodiments, the term "cycloalkyl" refers to a monocyclic ring system, a bicyclic ring system, or a polycyclic cycloalkyl ring system. In embodiments, a monocyclic ring system is a cyclic hydrocarbon group containing from 3 to 8 carbon atoms, wherein such groups may be saturated or unsaturated, but not aromatic. In embodiments, a cycloalkyl group is fully saturated. A bicyclic ring system or a polycyclic cycloalkyl ring system refers to multiple rings fused together, wherein at least one of the fused rings is a cycloalkyl ring, and wherein the multiple rings are attached to the parent molecular moiety through any carbon atom contained within the cycloalkyl rings of the multiple rings.

[0058] In an embodiment, the cycloalkyl group is a cycloalkenyl group. The term "cycloalkenyl" is used according to its plain ordinary meaning. In an embodiment, the cycloalkenyl group is a monocyclic ring system, a bicyclic ring system, or a polycyclic cycloalkenyl ring system. A bicyclic ring system or a polycyclic cycloalkenyl ring system refers to a plurality of rings fused together, wherein at least one of the fused rings is a cycloalkenyl ring, and wherein the plurality of rings is attached to the parent molecular moiety through any carbon atom contained within the cycloalkenyl rings of the plurality of rings.

[0059] In embodiments, the term "heterocycloalkyl" means a monocyclic ring system, a bicyclic ring system, or a polycyclic heterocycloalkyl ring system. In embodiments, the heterocycloalkyl is fully saturated. A bicyclic ring system or a polycyclic heterocycloalkyl ring system refers to multiple rings fused together, wherein at least one of the fused rings is a heterocycloalkyl ring, and wherein the multiple rings are attached to the parent molecular moiety through any atom contained within the heterocycloalkyl rings of the multiple rings.

[0060] Unless otherwise stated, the term "halogen" or "halogen," alone or as part of another substituent, means a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl" are intended to include both monohaloalkyl and polyhaloalkyl. For example, the term "halo(C1-C4)alkyl" includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, etc.

[0061] Unless otherwise stated, the term "acyl" means -C(O)R, where R is a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heteroalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl.

[0062] Unless otherwise stated, the term "aryl" refers to a polyunsaturated aromatic hydrocarbon substituent, which may be a monocyclic or fused together (i.e., fused-ring aryl) or covalently linked multiple rings (preferably 1 to 3 rings). A fused-ring aryl refers to multiple fused rings, wherein at least one of the fused rings is an aryl ring, and wherein the multiple rings are connected to a parent molecule moiety via any carbon atom contained within the aryl ring of the multiple rings. The term "heteroaryl" refers to an aryl (or ring) containing at least one heteroatom, such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized. Thus, the term "heteroaryl" includes fused-ring heteroaryl (i.e., multiple fused rings, wherein at least one of the fused rings is a heteroaryl ring, and wherein the multiple rings are connected to a parent molecule moiety via any atom contained within the heteroaryl ring of the multiple rings). 5,6-fused-ring heteroaryl refers to two fused rings, one ring having 5 members and the other ring having 6 members, and wherein at least one ring is a heteroaryl ring. Similarly, 6,6-fused-ring heteroaryl refers to two fused rings, one with 6 members and the other with 6 members, and at least one of the rings is a heteroaryl ring. And 6,5-fused-ring heteroaryl refers to two fused rings, one with 6 members and the other with 5 members, and at least one of the rings is a heteroaryl ring. The heteroaryl group can be attached to the rest of the molecule via a carbon atom or a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furanyl, thiopheneyl, pyridinyl, pyrimidinyl, benzothiazolyl, benzoxylbenzimidazolyl, benzofuranyl, isobenzofuranyl, indolyl, isoindolyl, benzothiapheneyl, isoquinolinyl, quinoxalinyl, quinolinyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl. Pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isooxazolyl, 4-isooxazolyl, 5-isooxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furanyl, 3-furanyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-benzothiazolyl, purinel, 2-benzimidazolyl, 5-indolyl, 1-isoquinolinyl, 5-isoquinolinyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolinyl, and 6-quinolinyl. The substituents in each of the aryl and heteroaryl ring systems mentioned above are selected from the group of acceptable substituents described below. "Arylene" and "heteroarylene" by themselves or as part of another substituent refer to a divalent radical derived from aryl and heteroaryl, respectively. A heteroaryl substituent may be -O-bonded to a ring heteroatom nitrogen.

[0063] Spirocycle is two or more rings, wherein adjacent rings are connected by a single atom. Each ring in the spirocycle can be the same or different. Each ring in the spirocycle can be substituted or unsubstituted, and can have a substituent different from each other ring in a group of spirocycles. The possible substituents of each ring in the spirocycle are possible substituents of the same ring when not being a part of the spirocycle (such as a substituent of a cycloalkyl ring or a heterocycloalkyl ring). The spirocycle can be substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene or substituted or unsubstituted heterocycloalkylene, and each ring in the spirocyclic group can be any one of the previously listed ones, including all rings with a type (for example, all rings are substituted heterocycloalkylene, wherein each ring can be the same or different substituted heterocycloalkylene). When referring to a spirocycle system, heterocycle spirocycle means spirocycle, wherein at least one ring is a heterocycle, and wherein each ring can be a different ring. When referring to a spirocycle system, the substituted spirocycle means that at least one ring is substituted, and each substituent can be optionally different.

[0064] symbol It indicates the connection point between the chemical part and the rest of the molecule or chemical formula.

[0065] As used in this article, the term "oxo" refers to oxygen bonded to a carbon atom by a double bond.

[0066] The term "alkylarylene" refers to an arylene moiety covalently bonded to an alkylene moiety (also referred to herein as an alkylene linker). In embodiments, the alkylarylene has the following formula:

[0067]

[0068] The alkylarylene moiety can be substituted on the alkylene moiety or the arylene linker, for example, at carbon 2, 3, 4, or 6, with halogen, oxo, -N, -CF, -CCl, -CBr, -CI, -CN, -CHO, -OH, -NH, -COOH, -CONH, -NO, -SH, -SOCH, -SOH, -OSOH, -SONH, -NHNH, -ONH, -NHC(O)NHNH, substituted or unsubstituted C1-C5 alkyl, or substituted or unsubstituted 2- to 5-membered heteroalkyl. In an embodiment, the alkylarylene group is unsubstituted.

[0069] Each of the above terms (e.g., "alkyl," "heteroalkyl," "cycloalkyl," "heterocycloalkyl," "aryl," and "heteroaryl") includes both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

[0070] Substituents for alkyl and heteroalkyl groups (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more groups selected from, but not limited to, the following groups: -OR', =O, =NR', =N-OR', -NR'R", -SR', halogen, -SiR'R"R"', -OC(O)R', -C(O)R', -C02R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'C(O)NR"R"', -N R″C(O)2R′, -NRC(NR′R″R″′)=NR″″, -NRC(NR′R″)=NR″, -S(O)R′, -S(O)2R′, -S(O)2NR′R″, -NRSO2R′, -NR′NR″R″′, -ONR′R″, -NR′C(O)NR″NR″′R″″, -CN, -NO2, -NR′SO2R″, -NR′C(O)R″, -NR′C(O)OR″, -NR′OR′′, in a number ranging from zero to (2m′+1), where m′ is the total number of carbon atoms in such a group. R', R", R"' and R"" each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, alkoxy or thioalkoxy, or arylalkyl. When a compound described herein contains more than one R group, for example, each R group is independently selected, as are each R', R", R"' and R"" group when more than one of these groups is present. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-membered ring, a 5-membered ring, a 6-membered ring, or a 7-membered ring. For example, -NR'R" includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one skilled in the art will understand that the term "alkyl" is intended to include groups that include carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl groups (e.g., -CF3 and -CH2CF3) and acyl groups (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, etc.).

[0071] Similar to the substituents described for alkyl, the substituents for aryl and heteroaryl are different and are selected from, for example: -OR', -NR'R", -SR', halogen, -SiR'R"R"', -OC(O)R', -C(O)R', -CO2R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'C(O)NR"R"', -NR"C(O)2R', -NR-C(N R′R″R″′)=NR″″, -NR-C(NR′R″)=NR″′, -S(O)R′, -S(O)2R′, -S(O)2NR′R″, -NRSO2R′, -NR′NR″R ″′, -ONR′R″, -NR′C(O)NR″NR″′R″″, -CN, -NO2, -R′, -N3, -CH(Ph)2, fluoro(C1-C4)alkoxy, and The number of fluoro(C1-C4)alkyl, -NR′SO2R″, -NR′C(O)R″, -NR′C(O)OR″, and -NR′OR″ ranges from zero to the total number of open valences on the aromatic ring system. Furthermore, R′, R″, R″′, and R″″ are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. When the compound described herein contains more than one R group, for example, each R group is selected independently, as are each R′, R″, R″′, and R″″ groups, when more than one of these groups is present.

[0072] Substituents in the ring (e.g., cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene) can be described as substituents on the ring, rather than on a specific atom of the ring (often referred to as floating substituents). In this case, the substituent can be attached to any ring atom (following the valence rules), and in the case of fused or spirocyclic rings, the substituent described as being associated with a member of the fused or spirocyclic ring (floating substituents on a monocyclic ring) can be any substituent on a fused or spirocyclic ring (floating substituents on a polycyclic ring). When a substituent is attached to the ring, but not to a specific atom (floating substituent), and the substituent's subscript is an integer greater than one, multiple substituents can be on the same atom, the same ring, different atoms, different fused rings, different spirocyclic rings, and each substituent can optionally be different. When the connection point between the ring and the rest of the molecule is not limited to a single atom (floating substituent), the connection point can be any atom of the ring, and in the case of fused or spirocyclic rings, it is any atom of any fused or spirocyclic ring, while following the valence rules. When a ring, fused ring, or spirocyclic ring contains one or more cyclic heteroatoms, and the ring, fused ring, or spirocyclic ring shows one or more floating substituents (including, but not limited to, connection points with the rest of the molecule), the floating substituents can bond to the heteroatoms. In structures or formulas with floating substituents, when the cyclic heteroatom is shown to be bonded to one or more hydrogen atoms (e.g., having two cyclic nitrogen atoms bonded to the ring atom and a third bonded to hydrogen), the substituents are understood to substitute for hydrogen atoms when the heteroatom is bonded to the floating substituents, while following the rules of chemical valence.

[0073] Two or more substituents may optionally be linked to form aryl, heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-called cyclizing substituents are typically, but not necessarily, linked to a cyclic base structure. In one embodiment, the cyclizing substituent is linked to an adjacent member of the base structure. For example, two cyclizing substituents linked to an adjacent member of a cyclic base structure produce a fused ring structure. In another embodiment, the cyclizing substituent is linked to a single member of the base structure. For example, two cyclizing substituents linked to a single member of a cyclic base structure produce a spirocyclic structure. In yet another embodiment, the cyclizing substituent is linked to a non-adjacent member of the base structure.

[0074] Two substituents on adjacent atoms of an aryl or heteroaryl ring may optionally form a group of the formula -TC(O)-(CRR') q -U-, wherein T and U are independently -NR-, -O-, -CRR'- or a single bond, and q is an integer from 0 to 3. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring may be optionally replaced by a substituent of the formula -A-(CH2) rThe substituents of -B- are replaced, where A and B are independently -CRR′-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR′-, or single bonds, and r is an integer from 1 to 4. One of the single bonds in the newly formed ring may optionally be replaced by a double bond. Alternatively, the two substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced by the formula -(CRR′). s -X′-(C″R″R″′) d - Substituent substitution, wherein s and d are independently integers from 0 to 3, and X′ is -O-, -NR′-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR′-. Substituents R, R′, R″, and R″′ are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

[0075] As used herein, the term "heteroatom" or "cyclic heteroatom" is intended to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), selenium (Se), and silicon (Si). In embodiments, the term "heteroatom" or "cyclic heteroatom" is intended to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0076] As used herein, "substituent" means a group selected from the following moieties:

[0077] (A) oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -OSO3H, -SO2NH2, -NHNH2, -ONH2, -N HC(O)NHNH2, -NHC(O)NH2, -NHC(NH)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -N3, -SF5, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C 10 Aryl, C 10 aryl or phenyl), or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), and

[0078] (B) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), aryl (e.g., C6-C 10 Aryl, C 10 aryl or phenyl), heteroaryl groups substituted with at least one of the following substituents (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl):

[0079] (i) Oxygenated, Halogenated, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -OSO3H, -SO2NH2, -NHNH2, -ONH2, -N HC(O)NHNH2, -NHC(O)NH2, -NHC(NH)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -N3, -SF5, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C 10 Aryl, C 10 aryl or phenyl), or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), and

[0080] (ii) Alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), aryl (e.g., C6-C... 10 Aryl, C 10 aryl or phenyl), heteroaryl groups substituted with at least one of the following substituents (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl):

[0081] (a) Oxygenated, Halogenated, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -OSO3H, -SO2NH2, -NHNH2, -ONH2, -N HC(O)NHNH2, -NHC(O)NH2, -NHC(NH)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -N3, -SF5, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C 10 Aryl, C 10 aryl or phenyl), or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), and

[0082] (b) Alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), aryl (e.g., C6-C... 10 Aryl, C 10-aryl or phenyl), heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), substituted with at least one substituent selected from the group consisting of oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCHI, -OCH2F, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -OS O3H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHC(NH)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -N3, -SF5, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C 10 Aryl, C 10 aryl or phenyl), or an unsubstituted heteroaryl (e.g., a 5- to 10-membered heteroaryl, a 5- to 9-membered heteroaryl, or a 5- to 6-membered heteroaryl).

[0083] As used herein, "restricted size substituent" or "restricted size substituent" means a group selected from all substituents above for "substituent", wherein each substituted or unsubstituted alkyl group is a substituted or unsubstituted C1-C 20 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2-20-membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3-8-membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C 10 aryl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5- to 10-membered heteroaryl.

[0084] As used herein, “lower substituent” or “lower substituent group” means a group selected from all substituents mentioned above for “substituent”, wherein each substituted or unsubstituted alkyl group is a substituted or unsubstituted C1-C8 alkyl group, each substituted or unsubstituted heteroalkyl group is a substituted or unsubstituted 2- to 8-membered heteroalkyl group, each substituted or unsubstituted cycloalkyl group is a substituted or unsubstituted C3- to 7-membered cycloalkyl group, each substituted or unsubstituted heterocycloalkyl group is a substituted or unsubstituted 3- to 7-membered heterocycloalkyl group, each substituted or unsubstituted aryl group is a substituted or unsubstituted phenyl group, and each substituted or unsubstituted heteroaryl group is a substituted or unsubstituted 5- to 6-membered heteroaryl group.

[0085] In some embodiments, each substituent described in the compounds herein is substituted with at least one substituent. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene described in the compounds herein is substituted with at least one substituent. In other embodiments, at least one or all of these groups are substituted with at least one size-restricted substituent. In other embodiments, at least one or all of these groups are substituted with at least one lower substituent.

[0086] In other embodiments of the compounds herein, each substituted or unsubstituted alkyl group may be a substituted or unsubstituted C1-C 20 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2-20-membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3-8-membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C 10 aryl, and / or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5- to 10-membered heteroaryl. In some embodiments of the compounds herein, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C 20 alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 20-membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C8 cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 8-membered heterocycloalkylene, each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C 10 arylene, and / or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5- to 10-membered heteroarylene.

[0087] In some embodiments, each substituted or unsubstituted alkyl group is a substituted or unsubstituted C1-C8 alkyl group, each substituted or unsubstituted heteroalkyl group is a substituted or unsubstituted 2- to 8-membered heteroalkyl group, each substituted or unsubstituted cycloalkyl group is a substituted or unsubstituted C3-C7 cycloalkyl group, each substituted or unsubstituted heterocycloalkyl group is a substituted or unsubstituted 3- to 7-membered heterocycloalkyl group, and each substituted or unsubstituted aryl group is a substituted or unsubstituted C6-C 10 In some embodiments, each substituted or unsubstituted alkylene group is a substituted or unsubstituted C1-C8 alkylene group, each substituted or unsubstituted heteroalkylene group is a substituted or unsubstituted 2-8 membered heteroalkylene group, each substituted or unsubstituted cycloalkylene group is a substituted or unsubstituted C3-C7 cycloalkylene group, each substituted or unsubstituted heterocycloalkylene group is a substituted or unsubstituted 3-7 membered heterocycloalkylene group, each substituted or unsubstituted arylene group is a substituted or unsubstituted C6-C 10 In some embodiments, the compound is a chemical species listed in the embodiments section, figures, or tables below.

[0088] In embodiments, the substituted or unsubstituted moiety (e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, and / or substituted or unsubstituted heteroarylene) is unsubstituted (e.g., unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted alkylene, unsubstituted heteroalkylene, unsubstituted cycloalkylene, unsubstituted heterocycloalkylene, unsubstituted arylene, and / or unsubstituted heteroarylene, respectively). In embodiments, the substituted or unsubstituted moiety (e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, and / or substituted or unsubstituted heteroarylene) is substituted (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene, respectively).

[0089] In embodiments, the substituted portion (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene) is substituted with at least one substituent, wherein if the substituted portion is substituted with multiple substituents, each substituent may optionally be different. In embodiments, if the substituted portion is substituted with multiple substituents, each substituent is different.

[0090] In embodiments, the substituted portion (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene) is substituted with at least one size-restricted substituent, wherein if the substituted portion is substituted with multiple size-restricted substituents, each size-restricted substituent may optionally be different. In embodiments, if the substituted portion is substituted with multiple size-restricted substituents, each size-restricted substituent is different.

[0091] In an embodiment, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene) is substituted with at least one lower substituent, wherein if the substituted moiety is substituted with multiple lower substituents, each lower substituent is optionally different. In an embodiment, if the substituted moiety is substituted with multiple lower substituents, each lower substituent is different.

[0092] In an embodiment, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene) is substituted with at least one substituent, size-restricted substituent, or lower substituent; wherein if the substituted moiety is substituted with a plurality of groups selected from substituents, size-restricted substituents, and lower substituents; each substituent, size-restricted substituent, and / or lower substituent is optionally different. In an embodiment, if the substituted moiety is substituted with a plurality of groups selected from substituents, size-restricted substituents, and lower substituents; each substituent, size-restricted substituent, and / or lower substituent is different.

[0093] Certain compounds of the present disclosure have asymmetric carbon atoms (optical or chiral centers) or double bonds; based on absolute stereochemistry, enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisomeric forms of amino acids can be defined as (R)- or (S)- or (D)- or (L)-, and each isomer is encompassed within the scope of the present disclosure. The compounds of the present disclosure do not include those known in the art that are too unstable to synthesize and / or separate. The present disclosure is intended to include compounds in racemic and optically pure forms. Optically active (R)- and (S)- or (D)- and (L)-isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic bonds or other centers of geometric asymmetry, and unless otherwise indicated, it is intended that the compounds include E and Z geometric isomers.

[0094] As used herein, the term "isomers" refers to compounds that have the same number and kind of atoms, and therefore the same molecular weight, but differ in the structural arrangement or configuration of the atoms.

[0095] As used herein, the term "tautomer" refers to one of two or more structural isomers that exists in equilibrium and is readily convertible from one isomer form to another.

[0096] It will be apparent to those skilled in the art that certain compounds of the present disclosure may exist in tautomeric forms, and all such tautomeric forms of the compounds are within the scope of the present disclosure.

[0097] Unless otherwise stated, structures depicted herein are also intended to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the compounds of the present invention are within the scope of the present disclosure.

[0098] Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, in addition to the replacement of hydrogen by deuterium or tritium, or by 13 C- or 14 Compounds of the present structures having C-enriched carbon substituted carbons are within the scope of this disclosure.

[0099] The compounds of the present disclosure may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as tritium ( 3 H), iodine-125 ( 125 i) or carbon-14( 14 C) All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are encompassed within the scope of the present disclosure.

[0100] It should be noted that throughout this application, candidates are written as Markush groups, e.g., each amino acid position contains more than one possible amino acid. It is specifically contemplated that each member of a Markush group should be considered individually, thereby comprising another embodiment, and a Markush group should not be construed as a single unit.

[0101] As used herein, the terms "bioconjugate" and "bioconjugate linker" refer to the binding between atoms or molecules of a bioconjugate reactive group or bioconjugate reactive moiety. The binding can be direct or indirect. For example, the conjugate between a first bioconjugate reactive group (e.g., -NH2, -COOH, -N-hydroxysuccinimide, or -maleimide) and a second bioconjugate reactive group (e.g., a sulfhydryl, a sulfur-containing amino acid, an amine, an amino acid with an amine side chain, or a carboxylate) provided herein can be direct, e.g., through a covalent bond or linker (e.g., a first linker to a second linker), or indirect, e.g., through a non-covalent bond (e.g., electrostatic interactions (e.g., ionic bonds, hydrogen bonds, halogen bonds), van der Waals interactions (e.g., dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (π effect), hydrophobic interactions, etc.). In embodiments, the bioconjugate or bioconjugate linker is formed using bioconjugate chemistry (i.e., the binding of two bioconjugate reactive groups), including but not limited to nucleophilic substitution (e.g., reaction of amines and alcohols with acyl halides, active esters), electrophilic substitution (e.g., enamine reaction), and carbon-carbon and carbon-heteroatom multiple bond additions (e.g., Michael reaction, Diels-Alder addition). These and other useful reactions are discussed in, for example, March, ADVANCED ORGANIC CHEMISTRY, 3rd ed., John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, D.C., 1982. In one embodiment, a first bioconjugation reactive group (e.g., a maleimide moiety) is covalently linked to a second bioconjugation reactive group (e.g., a thiol group). In another embodiment, a first bioconjugation reactive group (e.g., a haloacetyl moiety) is covalently linked to a second bioconjugation reactive group (e.g., a thiol group). In yet another embodiment, a first bioconjugation reactive group (e.g., an -N-hydroxysuccinimide moiety) is covalently linked to a second bioconjugation reactive group (e.g., an amine group). In yet another embodiment, a first bioconjugation reactive group (e.g., a maleimide moiety) is covalently linked to a second bioconjugation reactive group (e.g., a thiol group).In embodiments, a first bioconjugate reactive group (eg, a -sulfo-N-hydroxysuccinimide moiety) is covalently linked to a second bioconjugate reactive group (eg, an amine).

[0102] Useful bioconjugation reactive components for the bioconjugation chemistry described herein include, for example: (a) carboxyl groups and their various derivatives, including but not limited to, N-hydroxysuccinimide esters, N-hydroxybenzotriazole esters, acyl halides, acyl imidazoles, thioesters, p-nitrobenzene esters, alkyl, alkenyl, alkynyl, and aromatic esters; (b) hydroxyl groups that can be converted into esters, ethers, aldehydes, etc.; (c) haloalkyl groups, wherein the halide may be subsequently substituted by a nucleophilic group, such as, for example, an amine, a carboxylate anion, a thiol anion, a carbanion, or an alkoxy ion, resulting in the new group being covalently attached to the site of the halogen atom; (d) dienophilic groups capable of participating in Diels-Alder reactions, such as, for example, maleimide or maleimide groups; (e) aldehyde or ketone groups, such that subsequent derivatization can be carried out by forming carbonyl derivatives, such as, for example, imines, hydrazones, ureas, or oximes, or by mechanisms such as Grignard addition or alkyllithium addition; (f) sulfonyl halide groups. (g) Thiol groups, which can be converted to disulfides, react with acyl halides, or bonded to metals such as gold, or react with maleimide; (h) amines or thiol groups (e.g., present in cysteine), which can be, for example, acylated, alkylated or oxidized; (i) alkenes, which can undergo, for example, cycloaddition, acylation, Michael addition, etc.; (j) epoxides, which can react with, for example, amines and hydroxyl compounds; (k) phosphoramides and other standard functional groups used in nucleic acid synthesis; (i) metal silicon oxide bonding; (m) metal bonding to reactive phosphorus groups (e.g., phosphine) to form, for example, phosphodiester bonds; (n) coupling azides to alkynes using copper-catalyzed cycloaddition click chemistry; and (o) biotin conjugates that can react with avidin or streptoavidin to form avidin-biotin complexes or streptoavidin-biotin complexes.

[0103] The bioconjugate reactive groups can be selected so that they do not participate in or interfere with the chemical stability of the conjugates described herein. Alternatively, the reactive functional groups can be protected from participating in cross-linking reactions by the presence of protecting groups. In embodiments, the bioconjugate comprises a molecular entity derived from the reaction of an unsaturated bond (such as maleimide) and a sulfhydryl group.

[0104] The terms "analog," "analogue," or "derivative" are used in their ordinary, common sense in chemistry and biology, and refer to a chemical compound that is structurally similar to another compound (the so-called "reference" compound) but has a different composition. For example, one atom may be substituted by an atom of a different element, or a specific functional group may be present, or one functional group may be substituted by another, or the reference compound may have one or more chiral centers with absolute stereochemistry. Therefore, an analog is a compound that is similar or equivalent to a reference compound in function and appearance, but differs from the reference compound in structure or origin.

[0105] As used herein, the term "a" or "an" means one or more. In addition, as used herein, the phrase "substituted" means that the specified group may be substituted with one or more of any or all of the specified substituents. For example, when a group such as an alkyl or heteroaryl group is "substituted with an unsubstituted C1-C 20 When alkyl or unsubstituted heteroalkyl groups of 2 to 20 members are substituted, the group may contain one or more unsubstituted C1-C groups. 20 alkyl and / or one or more unsubstituted 2- to 20-membered heteroalkyl groups.

[0106] Furthermore, when a moiety is substituted with an R substituent, the group may be referred to as "R-substituted." When a moiety is R-substituted, the moiety is substituted with at least one R substituent, and each R substituent is optionally different. Where a particular R group is present in the description of a chemical genus, such as Formula (I), each occurrence of that particular R group may be distinguished by a Roman letter symbol. For example, when multiple R groups are present, 13 When a substituent is present, each R 13 Substituents can be divided into R 13.A 、R 13.B 、R 13.C 、R 13.D etc., where each R 13.A 、R 13.B 、R 13.C 、R 13.D Waiting in R 13 When an R moiety, group, or substituent as disclosed herein is attached by a single bond, and the R moiety, group, or substituent is an oxo, one of ordinary skill in the art will immediately recognize that, according to the normal rules of chemical valence, the oxo is attached by a double bond.

[0107] The description of the compounds disclosed herein is limited by principles of chemical bonding known to those skilled in the art. Therefore, when a group can be substituted by one or more of a plurality of substituents, such substitution is chosen to conform to the principles of chemical bonding and to produce compounds that are not inherently unstable and / or known to those skilled in the art to be potentially unstable under environmental conditions (such as aqueous, neutral, and several known physiological conditions). For example, according to principles of chemical bonding known to those skilled in the art, heterocyclic alkyl or heteroaryl groups are linked to the remainder of the molecule via cyclic heteroatoms, thereby avoiding inherently unstable compounds.

[0108] In the context of nucleic acid probes and target nucleic acids, the term "specifically interacting" (and similar terms) indicates that the probe is designed to interact with the target to a greater extent than with non-target nucleic acids also present in the reaction mixture. For example, specific interaction may include the probe hybridizing to the corresponding target in whole or in part. Hybridization between the probe and the target does not need to be 100%. For example, functionally effective interaction can be achieved with probes that have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at most 100% homology with their corresponding targets.

[0109] As used herein, a “detection channel” is a specified subset of the total range of possible values ​​for a detectable signal. For example, in the case where the detectable signal is a fluorescent signal, a detection channel (i.e., a fluorescent channel or dye channel) may represent a wavelength band of a specified size. A detection channel may, for example, have a band size of approximately 10 nm–60 nm, depending on instrument characteristics such as sensitivity and / or desired signal granularity. Detection channels may also include discontinuous wavelengths or wavelength ranges. Detection channels may be additionally or alternatively defined depending on the filter arrangement used to measure the detectable signal. Each distinct detection channel typically includes a specific filter arrangement to block non-channel emission. Thus, as a functional definition, each detectable signal within a given filter arrangement can be considered to be within the same detection channel. In some cases, different fluorescent labels (e.g., different chemical structures) are still detected using the same detection channel. For example, the fluorescent dyes Cy5 and Alexa647 provide similar emission wavelengths and can be detected within the same channel.

[0110] As used herein, “substantially identical” signals are signals that cannot be clearly distinguished from each other under the detection conditions used. Optionally, the emission spectra of two substantially identical signals overlap to the extent that each signal cannot be detected individually, such as a composite emission spectrum that does not show the presence of two distinct peaks. Optionally, “substantially identical” fluorescence emission may be within similar wavelength bands. For example, a first fluorescence signal and a second fluorescence signal having substantially identical fluorescence may have emission peaks that differ by no more than about 10 nm, or no more than about 8 nm, or no more than about 6 nm, or no more than about 4 nm, or no more than about 2 nm, or no more than about 1 nm, or are substantially indistinguishable from each other by detection instruments used to measure fluorescence emission. Additionally, or alternatively, in applications using the same detection apparatus, such as the same filter arrangement, to measure fluorescence signals, fluorescence signals may be considered to have “substantially identical fluorescence.” In embodiments, substantially identical signals have substantially identical excitation / absorption spectra, such that they cannot be excited individually. Optionally, both tags are excited during detection. The two tags may be excited and / or detected simultaneously.

[0111] As used herein with respect to a signal, “substantial” indicates a level significantly above the background. For example, a “substantial signal” and / or a detectable signal with “substantial fluorescence” is a signal significantly above the background (i.e., baseline) level, including fluorescence signals significantly above the fluorescence background / baseline level. This can be defined by a threshold that distinguishes background fluorescence from substantial fluorescence. The threshold can vary depending on the specific testing protocol and application requirements. In some embodiments (e.g., without a passive reference), the threshold is set to, for example, a ΔRn of about 1,000 to about 30,000, or about 2,000 to about 20,000, or about 3,000 to about 15,000, or about 4,000 to about 6,000, or within a range having any two endpoints defined by the foregoing values. In some embodiments (e.g., using a passive reference), the threshold is set to, for example, a ΔRn of about 0.01 to 0.5. In some embodiments, the threshold is higher than the baseline level by some percentage, such as about 5% to about 10% above the baseline level.

[0112] The “background” or “baseline” level of the signal during the amplification process (i.e., the background / baseline level of fluorescence) can be determined according to methods known to those skilled in the art. As a non-limiting example, the baseline level can be determined as the median signal of the amplification cycle prior to exponential amplification. For example, exponential amplification can be identified when the signal change from one amplification cycle to the next exceeds a certain percentage indicating an exponential change.

[0113] As a corollary, a signal and / or fluorescence level that is not "substantial" according to the foregoing may be described herein as "negligible." Similarly, a probe is "substantially bound" to a target when the probe is significantly above background (e.g., above binding to non-targets) relative to probe binding. Optionally, at least 1%, 5%, 10%, 20%, 50%, or 80% of the probe or target is bound.

[0114] As used herein, a "cleavable" probe is one that is intended to be cleaved as a result of the specific interaction of the probe with its corresponding target, thereby resulting in the release of the corresponding label and an increase in the corresponding detectable signal.

[0115] As used herein, a "non-cleavable" probe is a probe having a label that is intended to remain associated with the probe throughout the assay. In a non-cleavable probe, the corresponding detectable signal changes based on changes in the probe's configuration, rather than based on the release of the label from the probe. Extendable fluorescent probes, such as universal or hairpin extendable fluorescent probes, as described in various embodiments, are examples of non-cleavable probes.

[0116] The terms "detectable signal" and "label signal" are used synonymously herein. For example, a "first label signal" is a signal emitted by a first label of a first probe type, and a "second label signal" is a signal emitted by a second label of a second probe type. A "total signal" is the total measured signal within a specific detection channel at a given time point or measurement point. A plurality of different "detectable signals" / "label signals" may contribute to the same "total signal". For example, the total signal may include a signal generated by a first label of a first probe type and a signal generated by a second label of a second probe type. In some embodiments, the signal is a fluorescent signal, and terms such as "first fluorescent signal", "second fluorescent signal", and "total fluorescent signal" may be used as specific examples of corresponding broader terms.

[0117] The terms "determine," "calculate," and "estimate" are used synonymously herein. These terms are not intended to imply a precise level of measurement accuracy. Thus, where values ​​are "determined," "calculated," or "estimated" using the embodiments described herein, it should be understood that such values ​​may include some degree of inherent error due to factors such as instrument tolerances, rounding, chemical reaction variability, and other inherent measurement imperfections known and understood by those skilled in the art.

[0118] For any given element of a component of a described embodiment, any possible alternatives listed for that element or component can generally be used alone or in combination with one another, unless implicitly or explicitly stated otherwise.

[0119] Furthermore, unless otherwise specified, the figures used in the specification and claims to express quantities, composition, distances, or other measures should be understood as optionally modified by the term "about" or its synonyms. When the terms "about," "approximately," "substantially," etc., are used in conjunction with the stated quantity, value, or condition, they may mean a quantity, value, or condition that deviates from the stated quantity, value, or condition by less than 20%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01%. To a minimum, and without attempting to limit the application of the equivalence principle to the scope of the claims, each numerical parameter should be interpreted based on the number of significant figures reported and by applying ordinary rounding techniques. In embodiments, "about" means within the standard deviation using measurements generally acceptable in the art. In embodiments, "about" means a range extending to + / - 10% of the specified value. In embodiments, "about" includes a specific value.

[0120] A "detectable agent," "detectable compound," "detectable label," or "detectable moiety" is a substance (e.g., an element), molecule, or composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, magnetic resonance imaging, or other physical means. For example, detectable agents include 18 F. 32 P. 33 P. 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu、 67 Cu, 67 Ga, 68 Ga, 77 As、 86 Y. 90 Y. 89 Sr. 89 Zr, 94 Tc, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag、 111 In, 123 I. 124 I. 125 I. 131 I. 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154-1581 Gd, 161 Tb, 166 Dy,166 Ho、 169 Second, 175 Lu, 177 Lu, 186 Re、 188 Re、 189 Re、 194 Ir, 198 Au、 199 Au、 211 At、 211 Pb, 212 Bi、 212 pb, 213 Bi、 223 Ra, 225 Ac, Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, 32 P, fluorophores (e.g., fluorescent dyes), modified oligonucleotides (e.g., portions described in PCT / US2015 / 022063, which are incorporated herein by reference), electron-dense reagents, enzymes (e.g., commonly used in ELISA), biotin, digoxigenin, paramagnetic molecules, paramagnetic nanoparticles, ultrasmall superparamagnetic iron oxide (“USPIO”) nanoparticles, USPIO nanoparticle aggregates, superparamagnetic iron oxide (“SPIO”) nanoparticles, SPIO nanoparticle aggregates, monoferric oxide nanoparticles, monoferric oxide, nanoparticle contrast agents, liposomes or molecules containing gadolinium chelates (“Gd-chelates”), gadolinium, radioisotopes, radionuclides (e.g., carbon-11, nitrogen-13, oxygen-15, fluorine-18, rubidium-82), fluorodeoxyglucose (e.g., fluorine-18 labeled), any emitting gamma rays Radionuclides, positron-emitting radionuclides, radiolabeled glucose, radiolabeled water, radiolabeled ammonia, biocolloids, other delivery media of microbubbles (e.g., including microbubble shells comprising albumin, galactose, lipids and / or polymers; microbubble cores comprising air, heavy gases, perfluorocarbon, nitrogen, octafluoropropane, perfluorohexane lipid microspheres, perfluoropropane, etc.), iodinated contrast agents (e.g., iohexol, iodixanol, ioflufenicol, iopamidol, iodixyllanth, iopromide, diatrizoate, methatrizoate, iodixanol), barium sulfate, thorium dioxide, gold, gold nanoparticles, gold nanoparticle aggregates, fluorophores, two-photon fluorophores, or haptens and proteins or other entities, which can be made detectable, for example, by incorporating a radiolabel into a peptide or antibody that specifically reacts with a target peptide. In embodiments, the detectable portion is a portion of the detectable agent (e.g., in monovalent form). In the implementation, the detectable marking portion is a portion of the detectable marking (e.g., in unit price form).

[0121] The terms "fluorophore" or "fluorescent agent" or "fluorescent dye" are used interchangeably and refer to a substance, compound, agent (e.g., a detectable agent), or composition (e.g., a compound) that absorbs one or more wavelengths of light and re-emit light at one or more longer wavelengths relative to the absorbed wavelength or wavelengths of light. Examples of fluorophores that can be included in the compounds and compositions described herein include fluorescent proteins, xanthene derivatives (e.g., fluorescein, rhodamine, oregano green, eosin, or Texas Red), cyanines and derivatives (e.g., cyanine, indocarbocyanine, oxycarbocyanine, thiocarbocyanine, or merocyanine), naphthalene derivatives (e.g., dansyl or prodan derivatives), coumarins and derivatives, oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole, or benzoxadiazole), anthracene derivatives (e.g., anthraquinone, DRAQ5, DRAQ7, or CyTRAKOrange) (e.g., proflavine, acridine orange, acridine yellow), pyrene derivatives (e.g., Cascade Blue and its derivatives), oxazine derivatives (e.g., Nile Red, Nile Blue, Cresyl Violet, or Oxazine 170), arylmethine derivatives (e.g., auramine, crystal violet, or malachite green), tetrapyrrole derivatives (e.g., porphine, phthalocyanine, bilirubin), CF dyes, TM 、DRAQ TM 、CyTRAK TM 、BODIPY TM Alexa Fluor TM 、DyLight Fluor TM 、Atto TM Tracy TM 、FluoProbes TM 、Abberior Dyes TM 、DY TM dyes、MegaStokes Dyes TM 、Sulfo Cy TM 、Seta TM Dye, SeTau TM Dyes, Square Dyes TM 、Quasar TM dyes、Cal Fluor TM Dyes, SureLightDyes TM 、PerCP TM 、Phycobilisomes TM 、APC TM 、APCXL TM , RPE TM and / or BPE TMThe fluorescent moiety is a functional group of the fluorescent agent. Emission from the fluorophore can be detected by any method, including but not limited to fluorescence spectroscopy, fluorescence microscopy, fluorometers, fluorescent plate readers, infrared scanner analysis, laser scanning confocal microscopy, automated confocal nanoscanning, laser spectrophotometers, fluorescence activated cell sorting systems (FACS), image-based analyzers, and fluorescence scanners (e.g., gel / membrane scanners). In one embodiment, the fluorophore is an aromatic (e.g., polyaromatic) moiety having a conjugated π-electron system. In another embodiment, the fluorophore is a fluorescent dye moiety, i.e., a monovalent fluorophore.

[0122] According to embodiments of this disclosure, radioactive materials (e.g., radioisotopes) that can be used as imaging agents and / or labeling agents include, but are not limited to, those that can be used as imaging agents and / or labeling agents. 18 F. 32 P. 33 P. 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 77 As、 86 Y. 90 Y. 89 Sr. 89 Zr, 94 Tc, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag、 111 In, 123 I. 124 I. 125 I. 131 I. 142 Pr, 143 Pr, 149 pm, 153 Sm, 154-1581 Gd, 161 Tb, 166 Dy, 166 Ho、 169 Second, 175 Lu, 177 Lu, 186 Re、 188 Re、 189 Re、 194 Ir, 198 Au、 199 Au、211 At 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra and 225 Ac. According to embodiments of this disclosure, paramagnetic ions that can be used as additional imaging agents include, but are not limited to, ions of transition metals and lanthanides (e.g., metals with atomic numbers 21-29, 42, 43, 44, or 57-71). These metals include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu.

[0123] Examples of detectable agents include imaging agents, including fluorescent and luminescent substances, molecules or compositions, including but not limited to a variety of organic or inorganic small molecules commonly referred to as "dyes", "labels" or "indicators". Examples include fluorescein, rhodamine, acridine dyes, Alexa dyes and cyanine dyes. In embodiments, the detectable portion is a fluorescent molecule (e.g., an acridine pigment, a cyanine dye, a fluoro dye, an oxazine dye, a phenanthridinium dye or a rhodamine dye). In embodiments, the detectable portion is a fluorescent molecule (e.g., an acridine pigment, a cyanine dye, a fluoro dye, an oxazine dye, a phenanthridinium dye or a rhodamine dye). In embodiments, the detectable portion is a fluorescent moiety or a fluorescent dye moiety. In embodiments, the detectable label is a fluorescent dye. In embodiments, the detectable label is a fluorescent dye that can exchange energy with another fluorescent dye (e.g., a fluorescence resonance energy transfer (FRET) chromophore).

[0124] As used herein, the term "quencher" refers to a substance, compound, agent, or composition (e.g., a compound) that is capable of absorbing energy from a detectable agent (e.g., a fluorophore or fluorescent dye) and re-emitting at least a portion of the energy (e.g., as heat (e.g., in the case of a dark quencher) or visible light (e.g., in the case of a fluorescence quencher)). In embodiments, the quencher absorbs energy from the fluorophore and re-emit at least a portion of the energy as heat in the case of a dark quencher. In embodiments, the quencher absorbs energy from the fluorophore and re-emit at least a portion of the energy as visible light in the case of a fluorescence quencher. A "quencher moiety" is a monovalent form of the quencher. An "internal quencher moiety" refers to a quencher moiety that is located at an internal site of the probe.

[0125] As used herein, the term "salt" refers to an acid or base salt of a compound described herein. Thus, the compounds of the present invention may exist as salts. The present invention includes such salts. Non-limiting examples of such salts include hydrochlorides, hydrobromides, phosphates, sulfates, methanesulfonates, nitrates, maleates, acetates, citrates, fumarates, propionates, tartrates (e.g., (-)-tartrates, (-)-tartrates, or mixtures thereof, including racemic mixtures), succinates, benzoates, and salts with amino acids such as glutamic acid, and quaternary ammonium salts (e.g., iodomethane, iodoethane, etc.). These salts can be prepared by methods known to those skilled in the art. Illustrative examples of acceptable salts are salts of inorganic acids (hydrochloric acid, hydrobromic acid, phosphoric acid, etc.), organic acids (acetic acid, propionic acid, glutamic acid, citric acid, etc.), and quaternary ammonium salts. In embodiments, the compound may have a positive charge, and it will be understood that appropriate counterions (e.g., chloride, fluoride, or acetate) may also be present, although not explicitly shown. Similarly, for compounds having a negative charge (e.g., ), it will be appreciated that appropriate counterions (e.g., protons, sodium ions, potassium ions, or ammonium ions) may also be present, even though not explicitly shown. The protonation state of a compound (e.g., a compound described herein) depends on the local environment (i.e., the pH of the environment). Thus, in embodiments, a compound may be described as having a protonated state (e.g., ) or ionic state (e.g. ) and it will be understood that these are interchangeable. In embodiments, the counterion is represented by the symbol A (e.g., A + or A - ).

[0126] The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.The parent form of the compound may differ from the various salt forms in certain physical properties, such as solubility in polar solvents.

[0127] Certain compounds of the present disclosure can exist in unsolvated forms as well as solvated forms (including hydrated forms). Generally, solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present disclosure. Certain compounds of the present disclosure can exist in polycrystalline or amorphous forms. Generally speaking, all physical forms are equivalent for the purposes contemplated by the present disclosure and are intended to be within the scope of the present disclosure.

[0128] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues, wherein the polymer may optionally be bound to a portion not composed of amino acids. These terms apply to amino acid polymers, where one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. A polypeptide or cell is “recombinant” when it is artificially or engineered, or derived from or contains artificially or engineered proteins or nucleic acids (e.g., non-natural or non-wild-type). For example, a polynucleotide inserted into a vector or any other heterologous site (e.g., in the genome of a recombinant organism) such that it does not associate with the nucleotide sequence normally side-joined to that polynucleotide is found in nature to be a recombinant polynucleotide. Proteins expressed in vitro or in vivo by recombinant polynucleotides are examples of recombinant polypeptides. Similarly, polynucleotide sequences not found in nature, such as variants of naturally occurring genes, are recombinant.

[0129] "Contact" is used in its ordinary, general sense and refers to a process that brings at least two different species (e.g., chemical compounds comprising biomolecules or cells) close enough to react, interact, or physically touch. However, it should be understood that the resulting reaction product may be generated directly from a reaction between the added reagents or from one or more intermediates from the added reagents that can be produced in the reaction mixture. The term "contact" can include allowing two species to react, interact, or physically touch, wherein the two species can be a compound and a protein or enzyme as described herein. In embodiments, contact includes allowing the compound described herein to interact with a protein or enzyme involved in a signal transduction pathway.

[0130] "Control" or "controlled experiment" is used in its ordinary sense and refers to an experiment in which the subjects or reagents are treated as in a parallel experiment, except that the experimental procedures, reagents, or variables are omitted. In some cases, controls are used as a comparative standard for evaluating the effects of an experiment.

[0131] The term "modulate" is used in its ordinary, common sense, referring to the act of changing or varying one or more properties. "Modulation" refers to the process of altering or varying one or more properties. For example, when applied to the effect of a modulator on a target protein, modulation means altering the properties or function of the target molecule or the amount of the target molecule by increasing or decreasing it.

[0132] "Nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) in single-stranded, double-stranded, or multi-stranded form, their polymers, or complementary compounds thereof; or nucleosides (e.g., deoxyribonucleosides or ribonucleosides). In embodiments, "nucleic acid" does not include nucleosides. The terms "polynucleotide," "oligonucleotide," etc., generally and conventionally refer to straight-chain nucleotide sequences. Oligonucleotides are typically about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50, or more nucleotides in length, and at most about 100 nucleotides. Nucleic acids and polynucleotides are polymers of any length, including longer lengths, such as 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. In some embodiments, the nucleic acids herein contain phosphodiester bonds. Other embodiments include nucleic acid analogs that may have alternating backbones, including, for example, aminophosphates, thiophosphates, dithiophosphates, or O-methylphosphoramide links (see Eckstein, *Oligonucleotides and Analogues: A Practical Approach*, Oxford University Press); and peptide nucleic acid backbones and links. Other similar nucleic acids include those with a positive backbone; nonionic backbones and nonribose backbones, including U.S. Patents 5,235,033 and 5,034,506 and Chapters 6 and 7, "Carbohydrate Modifications in Antisense Research," of ASC Symposium Series 580, edited by Sanghui and Cook. Nucleic acids containing one or more carbocyclic sugars are also included in one definition of nucleic acids. Modification of the ribose-phosphate backbone can be for a variety of reasons, such as increasing the stability and half-life of such molecules in physiological environments or as probes on biochips. Mixtures of naturally occurring nucleic acids and analogues can be prepared; alternatively, mixtures of different nucleic acid analogues, as well as mixtures of naturally occurring nucleic acids and analogues, can be prepared. As described herein, the residues of nucleic acids are monomers of nucleic acids (e.g., nucleotides). The term "nucleoside" in its usual and conventional sense refers to a glycosylamine comprising a nucleobase and a pentose sugar (ribose or deoxyribose). Non-limiting examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine, and inosine. Nucleosides may be modified at the bases and / or sugars. The term "nucleotide" in its usual and conventional sense refers to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms thereof.Examples of polynucleotides covered herein include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, and hybrid molecules having mixtures of single-stranded and double-stranded DNA and RNA. Examples of nucleic acids, such as the polynucleotides contemplated herein, include any type of RNA, such as mRNA, siRNA, miRNA, and guide RNA, and any type of DNA, genomic DNA, plasmid DNA, and small circular DNA, and any fragments thereof. In the context of polynucleotides, the term “double-stranded” refers to double strands in the usual and conventional sense. Nucleic acids can be straight or branched. For example, a nucleic acid can be a straight chain of nucleotides, or a nucleic acid can be branched, such that the nucleic acid contains one or more arms or branches of nucleotides. Optionally, branched nucleic acids are repeatedly branched to form higher-order structures, such as dendritic polymers, etc. As used herein, a “nucleic acid moiety” is a monovalent form of a nucleic acid. In embodiments, the nucleic acid moiety is attached to the 3′ or 5′ position of a nucleotide or nucleoside.

[0133] Nucleic acids, including, for example, nucleic acids having a thiophosphate backbone, may include one or more reactive moieties. As used herein, the term reactive moiety includes any group capable of reacting with another molecule, such as a nucleic acid or polypeptide, through covalent, non-covalent, or other interactions. For example, nucleic acids may include amino acid reactive moieties that react with amino acids on a protein or polypeptide through covalent, non-covalent, or other interactions.

[0134] As used herein, "nucleotide" refers to a nucleoside-5'-phosphate (e.g., polyphosphate) compound or its structural analogues, which can be incorporated by a nucleic acid polymerase (e.g., partially incorporated as a nucleoside-5'-monophosphate or a derivative thereof) to extend a growing nucleic acid chain (e.g., a primer). Nucleotides may contain bases such as adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analogues thereof, and may contain 1, 2, 3, 4, 5, 6, 7, 8, or more phosphate groups. Nucleotides may be modified at one or more of the base, sugar, or phosphate groups. Nucleotides may be tagged or labeled ("tagged nucleotide" or "labeled nucleotide"). In embodiments, the nucleotide is a deoxyribonucleotide. In embodiments, the nucleotide is a ribonucleotide. In embodiments, the nucleotide contains three phosphate groups (e.g., triphosphate groups).

[0135] The term also includes nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that are synthetic, naturally occurring, and non-naturally occurring, with binding properties similar to reference nucleic acids, and metabolized in a manner similar to reference nucleotides. Examples of such analogs include, but are not limited to, phosphodiester derivatives, including, for example, phosphoramidates, phosphorodiaminoates, phosphorothioates (also known as phosphorothioates having double-bonded sulfur substituted for the oxygen in the phosphate), phosphorodithioates, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid, methylphosphonate, borophosphonate, or O-methylphosphoramidate linkages (see Eckstein, Oligonucleotides and Analogs: A Practical Approach, Oxford University Press), and modifications to nucleotide bases, such as 5-methylcytidine or pseudouridine; and peptide nucleic acid backbones and linkages. Other similar nucleic acids include those with positive backbones; non-ionic backbones, modified sugars, and non-ribose backbones (e.g., diaminophosphorothioate morpholino oligonucleotides or locked nucleic acids (LNA)), including those described in U.S. Patents 5,235,033 and 5,034,506 and Chapters 6 and 7 of ASC Seminar Series 580, "Carbohydrate Modifications in Antisense Research," eds. Sanghui and Cook. Nucleic acids containing one or more carbocyclic sugars are also included in one definition of nucleic acid. Modifications to the ribose-phosphate backbone can be for a variety of reasons, such as increasing the stability and half-life of such molecules in a physiological environment or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be prepared; alternatively, mixtures of different nucleic acid analogs can be prepared, as well as mixtures of naturally occurring nucleic acids and analogs. In an embodiment, the internucleotide linkage in the DNA is a phosphodiester, a phosphodiester derivative, or a combination of the two.

[0136] " Nucleoside " is similar in structure to nucleotide, but lacks the phosphate moiety present in nucleotide. The example of nucleoside analog is a nucleoside analog in which the label is connected to the base and there is no phosphate group connected to the sugar molecule. " Nucleoside " as used herein refers to a glycosyl compound consisting of a core base and a 5-membered ring sugar (e.g., ribose or deoxyribose). Nucleoside can include a base, such as adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U) or its analog. Nucleoside can be modified at the base and / or sugar. In an embodiment, the nucleoside is a deoxyribonucleoside. In an embodiment, the nucleoside is a ribonucleoside.

[0137] As used herein, the terms “complementary” or “fundamentally complementary” refer to hybridization, base pairing, or double-strand formation between nucleotides or nucleic acids. For example, complementarity exists between the two strands of a double-stranded DNA molecule or between primer binding sites on an oligonucleotide primer and a single-stranded nucleic acid when a nucleotide (e.g., RNA or DNA) or nucleotide sequence is capable of base pairing with a corresponding homologous nucleotide or homologous nucleotide sequence. As described herein and known in the art, the complementary (matching) nucleotide of adenosine (A) is thymidine (T), and the complementary (matching) nucleotide of guanosine (G) is cytosine (C). Therefore, a complement may comprise a nucleotide sequence that pairs with the corresponding complementary nucleotide of a second nucleic acid sequence. The nucleotides of the complement may partially or completely match the nucleotides of the second nucleic acid sequence. When the nucleotides of the complement completely match each nucleotide of the second nucleic acid sequence, the complement forms a base pair with each nucleotide of the second nucleic acid sequence. When the nucleotides of the complement partially match the nucleotides of the second nucleic acid sequence, only some of the nucleotides of the complement form a base pair with the nucleotides of the second nucleic acid sequence. Examples of complementary sequences include coding and non-coding sequences, where the non-coding sequence contains a nucleotide complementary to the coding sequence, and thus forms a complement of the coding sequence. Further examples of complementary sequences are sense and antisense sequences, where the sense sequence contains a complementary nucleotide to the antisense sequence, and thus forms a complement of the antisense sequence. "Double" means that at least two oligonucleotides and / or polynucleotides that are completely or partially complementary undergo Watson-Crick type base pairing in all or most of their nucleotides, thereby forming a stable complex.

[0138] As described herein, sequence complementarity can be partial, where only some nucleic acids in the nucleic acid match according to base pairing, or complete, where all nucleic acids match according to base pairing. Therefore, two sequences complementary to each other can have a specified percentage of nucleotides that are complementary to each other (e.g., about 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher complementarity in a specific region). In embodiments, when two sequences are completely complementary, they are complementary and have 100% complementarity.

[0139] As used herein, the term "polymerase" refers to any naturally occurring or non-naturally occurring enzyme or other catalyst capable of catalyzing polymerization reactions, such as the polymerization of nucleotide monomers to form nucleic acid polymers. Exemplary types of polymerases that can be used in the compositions and methods of this disclosure include nucleic acid polymerases, such as DNA polymerases, DNA- or RNA-dependent RNA polymerases, and reverse transcriptases. In some cases, the DNA polymerase is 9N polymerase or a variant thereof, E. coli DNA polymerase I, bacteriophage T4 DNA polymerase, sequencing enzyme, Taq DNA polymerase, DNA polymerase from Bacillus stearothermophilus, Bst 2.0 DNA polymerase, 9N polymerase, 9N polymerase (exonuclease-) A485L / Y409V, Phi29 DNA polymerase ( DNA polymerase), T7 DNA polymerase, DNA polymerase II, DNA polymerase III holoenzyme, DNA polymerase IV, DNA polymerase V, VentR DNA polymerase, Therminator TM II DNA polymerase, Therminator TM IIIDNA Polymerase or Therminator TMIX DNA polymerase. In embodiments, the polymerase is a protein polymerase. As used herein, the terms "DNA polymerase" and "nucleic acid polymerase" are used according to their ordinary meanings and refer to enzymes capable of synthesizing nucleic acid molecules from nucleotides (e.g., deoxyribonucleotides). Typically, a DNA polymerase adds nucleotides to the 3' end of a DNA chain, one nucleotide at a time. In this embodiment, the DNA polymerase is Pol I DNA polymerase, Pol II DNA polymerase, Pol III DNA polymerase, Pol IV DNA polymerase, Pol V DNA polymerase, Pol β DNA polymerase, Pol μ DNA polymerase, Pol λ DNA polymerase, Pol σ DNA polymerase, Pol α DNA polymerase, Pol δ DNA polymerase, Pol ε DNA polymerase, Pol η DNA polymerase, Pol ι DNA polymerase, Pol κ DNA polymerase, Pol ζ DNA polymerase, Pol γ DNA polymerase, Pol θ DNA polymerase, Pol υ DNA polymerase, or a thermophilic nucleic acid polymerase (e.g., thermal polymerase γ, 9N polymerase (exonuclease-), thermal polymerase II, thermal polymerase III, or thermal polymerase IX). In this embodiment, the DNA polymerase is a modified archaea DNA polymerase. In this embodiment, the polymerase is a reverse transcriptase. In embodiments, the polymerase is a mutant P. abyssi polymerase (e.g., such as a mutant P. abyssi polymerase described in WO 2018 / 148723 or WO 2020 / 056044). As used herein, the term "thermophilic nucleic acid polymerase" refers to a family of DNA polymerases (e.g., 9N TM) and its mutants, derived from the DNA polymerase originally isolated from the hyperthermophilic archaea (Thermococcus sp.) at 9 degrees N-7, found in hydrothermal vents at this latitude (Eastern Pacific Rise) (Southworth MW et al., Proceedings of the National Academy of Sciences (PNAS) 1996; 93(11): 5281-5285). Thermophilic nucleic acid polymerase is a member of B DNA polymerase. Site-directed mutagenesis of the 3′-5′ exonuclease I (Asp-Ile-Glu or DIE) into AIA, AIE, EIE, EID, or DIA produces a polymerase without detectable 3′ exonuclease activity. Mutation to Asp-Ile-Asp (DID) results in a 3′-5′ exonuclease specific activity reduced to <1% of the wild type, while retaining other properties of the polymerase, including its high chain substitution activity. Selected sequences AIA (D141A, E143A) are used to reduce exonuclease activity. Subsequent mutagenesis of key amino acids leads to an increased ability of the enzyme to incorporate dideoxynucleotides, ribonucleotides, and acyclic nucleotides (e.g., Therminator II enzyme from New England Biolabs with the D141A / E143A / Y409V / A485L mutation); 3′-amino-dNTPs, 3′-azido-dNTPs, and other 3′-modified nucleotides (e.g., NEB Therminator III DNA polymerase with the D141A / E143A / L408S / Y409A / P410V mutation, NEB Therminator IX DNA polymerase); or γ-phosphate-labeled nucleotides (e.g., Therminatorγ: D141A / E143A / W355A / L408W / R460A / Q461S / K464E / D480V / R484W / A485L). Typically, these enzymes do not possess 5'-3' exonuclease activity.Additional information on thermophilic nucleic acid polymerases can be found in the following references (Southworth MW et al. PNAS 1996;93(11):5281-5285; Bergen K et al. ChemBioChem 2013;14(9):1058-1062; Kumar S et al. Scientific Reports 2012;2:684; Fuller CW et al. 2016;113(19):5233-5238; Guo J et al. Proceedings of the National Academy of Sciences of the United States of America 2008;105(27):9145-9150), which are incorporated herein in their entirety for all purposes.

[0140] As used herein, the term "exonuclease activity" is used according to its ordinary meaning in the art, and refers to the removal of nucleotides from nucleic acids by DNA polymerase. For example, during polymerization, nucleotides are added to the 3' end of the primer chain. Sometimes DNA polymerase incorporates incorrect nucleotides at the 3'-OH end of the primer chain, wherein the incorrect nucleotide cannot form a hydrogen bond with the corresponding base in the template chain. Due to the 3' to 5' exonuclease activity of the DNA polymerase, the nucleotides added by such errors are removed from the primer. When referring to 3'-5' exonuclease activity, it is understood that the DNA polymerase promotes a hydrolysis reaction that destroys the phosphodiester bond at the 3' end of the polynucleotide chain to excise the nucleotides. In an embodiment, 3'-5' exonuclease activity refers to the continuous removal of nucleotides in single-stranded DNA in the 3'→5' direction, releasing deoxyribonucleoside 5'-monophosphates one by one. Methods for quantifying exonuclease activity are known in the art; see, for example, Southworth et al., Proc. Natl. Acad. Sci. USA 93, 8281-8285 (1996).

[0141] The term "endpoint" as used in reference to cycles is a designated cycle at which the PCR process is assumed to be complete and / or a designated cycle at which a signal threshold value above a specified amount of background signal appears. In various embodiments, the endpoint cycles according to the present disclosure may be in the range of 20 to 45 cycles, such as 30 to 40 cycles. However, the number of cycles to the endpoint cycle may vary. For example, the number of cycles to the endpoint cycle may be related to the position at which the emission (e.g., fluorescence) fluorescent signal indicating the amplification product reaches an approximate plateau. An "endpoint signal" refers to the emission signal measured during the endpoint cycle.

[0142] The term "spectral similarity" refers to the emission signals of detectable labels having the same spectral profile or substantially overlapping spectral profiles. Thus, different probe types carrying the same detectable label or different probe types carrying different detectable labels that have substantial spectral overlap in their emission signals can be considered probes with spectral similarity. In some embodiments, detectable labels with spectral similarity can be detected in the same optical detection channel, but other techniques can also be used to detect the emission signals of such detectable labels. Reference to substantially overlapping spectra should be understood to mean spectral similarity.

[0143] II.Compounds

[0144] In one aspect, compounds or salts thereof having the following formula are provided:

[0145]

[0146] Q A It's the quencher part.

[0147] B is a divalent nucleobase.

[0148] L 1 It is a divalent linker.

[0149] R 2 Is it hydrogen or -OR 2A .

[0150] R 3 Yes-OR 3A or -OP(NR 3B R 3C )-OR 3A .

[0151] R 4 is hydrogen or unsubstituted methyl, or R 2 and R 4 The substituents are linked to form a substituted or unsubstituted heterocycloalkyl group (e.g., 3 to 8-membered, 3 to 6-membered, 4 to 6-membered, 4 to 5-membered, or 5 to 6-membered),

[0152] R 5 Yes-OR 5A .

[0153] R 2A 、R 3A 、R 3B 、R 3C and R 5AIndependently, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2 I, -OCH2F, substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4, or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2 to 8 members, 2 to 6 members, 4 to 6 members, 2 to 3 members, or 4 to 5 members), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6, or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8 members, 3 to 6 members, 4 to 6 members, 4 to 5 members, or 5 to 6 members), substituted or unsubstituted aryl (e.g., C6-C 10 or phenyl), or a substituted or unsubstituted heteroaryl (e.g., 5- to 10-membered, 5- to 9-membered, or 5- to 6-membered).

[0154] In the implementation plan, the quencher component is in the form of QSY7 at a single price. In the implementation plan, the quencher component is... In an embodiment, the quencher moiety is In an embodiment, the quencher moiety is a monovalent form of QSY21. In an embodiment, the quencher moiety is The unit price is in the form of [price]. In the implementation plan, the quencher portion is [priced in the form of a unit price]. In the implementation plan, the quencher component is in the monovalent form of QSY9. In the implementation plan, the quencher component is... In an embodiment, the quencher moiety is In the implementation scheme, the quencher component is in the unit form of BHQ1. In the implementation scheme, the quencher component is... The unit price is in the form of [price]. In the implementation plan, the quencher portion is [priced in the form of a unit price]. In the implementation scheme, the quencher component is in the unit form of BHQ2. In the implementation scheme, the quencher component is... The unit price is in the form of [price]. In the implementation plan, the quencher portion is [priced in the form of a unit price]. In an embodiment, the quencher moiety is a monovalent form of BHQ3. In an embodiment, the quencher moiety is In an embodiment, the quencher moiety is In the implementation scheme, the quencher portion is in monovalent form of Dabcyl. In the implementation scheme, the quencher portion is... The unit price is in the form of [price]. In the implementation plan, the quencher portion is [priced in the form of a unit price]. In an embodiment, the quencher moiety is a monovalent form of Dabsyl. In an embodiment, the quencher moiety is The unit price is in the form of Eclipse. In the implementation scheme, the quencher portion is in the form of Eclipse. In the implementation scheme, the quencher portion is... The unit price is in the form of [price]. In the implementation plan, the quencher portion is [priced in the form of a unit price]. In the implementation plan, the quencher portion is in the form of BBQ-650 at unit price. In the implementation plan, the quencher portion is... In embodiments, the quencher moiety is a monovalent form of Iowa Black RQ. In embodiments, the quencher moiety is a monovalent form of Iowa Black FQ. In embodiments, the above quencher moieties are all interchangeable. In embodiments, the quencher moiety can be in formula (I), (IA), (II), (III), (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (VI-4), (VI-5), (VII), (VII-1), (VII-2), (VII-3), (VII-4), (VII-5), (VIII), (VIII-1), (VIII-2), (VIII-3), (VIII-4), (VIII-5), (IX), (IX-1), (IX-2), (IX-3), (IX-4), (IX-5), (X), (XI), ( XII), (XIII), (XIV), (XV), (XV-1), (XV-2), (XV-3), (XV-4), (XV-5), (XVI), (XVI-1), (XVI-2), (XVI-3), (XVI-4), (XVI-5), (XVII), (XVII-1), (XVII-2), (XVII-3), (XVII-4), (XVII-5), (XVIII), (XVIII-1), (XVIII-2), (XVIII-3), (XVIII-4), (XVIII-5) and (XIX), and embodiments thereof, are substituted.

[0155] In one aspect, compounds or salts thereof having the following formula are provided:

[0156]

[0157] B is a divalent nucleobase.

[0158] L 1 It is a divalent linker.

[0159] R 2 Is it hydrogen or -OR 2A .

[0160] R 3 Yes-OR 3A or -OP(NR 3B R 3C )-OR 3A .

[0161] R 4 is hydrogen or unsubstituted methyl, or R 2 and R 4 The substituents are linked to form a substituted or unsubstituted heterocycloalkyl group (e.g., 3 to 8-membered, 3 to 6-membered, 4 to 6-membered, 4 to 5-membered, or 5 to 6-membered),

[0162] R 5 Yes-OR 5A .

[0163] R 1 and R 10 and -C1-C8, -C1-C6, -C1-C4, or -C1-C2. In some embodiments, the alkyl radicals are hydrogen, -CCl, -CBr, -CF, -CI, -CHCl, -CHBr, -CHF, -CHI, -CHCl, -CHBr, -CHF, -CHI, -CN, -OH, -NH, -COOH, -CONH, -OCCl, -OCF, -OCBr, -OCI, -OCHCl, -OCHBr, -OCHI, -OCHF, -OCHCl, -OCHBr, -OCHI, -OCHF, substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4, or C1-C2), or substituted or unsubstituted heteroalkyl (e.g., 2-8, 2-6, 4-6, 2-3, or 4-5 members).

[0164] R 6 、R 7 、R 8 and R 9 Independently, I am hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3R A-SO2NH2, -NHNH2, -ONH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCBr3, -OCF3, -OCI3, -OCH2Cl, -OCH2Br, -OCH2F, -OCH2I, -OCHCl2, -OCHBr2, -OCHF2, -OCHI2, -SF5, -N3, substituted or unsubstituted Alkyl groups (e.g., C1-C8, C1-C6, C1-C4, or C1-C2), substituted or unsubstituted heteroalkyl groups (e.g., 2 to 8-membered, 2 to 6-membered, 4 to 6-membered, 2 to 3-membered, or 4 to 5-membered), substituted or unsubstituted cycloalkyl groups (e.g., C3-C8, C3-C6, C4-C6, or C5-C6), substituted or unsubstituted heterocycloalkyl groups (e.g., 3 to 8-membered, 3 to 6-membered, 4 to 6-membered, 4 to 5-membered, or 5 to 6-membered), substituted or unsubstituted aryl groups (e.g., C6-C4, C1 to C2), substituted or unsubstituted heterocycloalkyl groups (e.g., 3 to 8-membered, 3 to 6-membered, 4 to 6-membered, 4 to 5-membered, or 5 to 6-membered), substituted or unsubstituted aryl groups (e.g., C6-C4, C1 to C2), substituted or unsubstituted alkyl groups (e.g., C1 ... to C4, C1 to C2), substituted or unsubstituted alkyl groups (e.g., C1 to C4, C1 to C2), substituted or unsubstituted alkyl groups (e.g., C1 to C4, C1 to C2), substituted or unsubstituted alkyl groups 10 or phenyl), or a substituted or unsubstituted heteroaryl (e.g., 5- to 10-membered, 5- to 9-membered, or 5- to 6-membered).

[0165] R 1 and R 6 They can be linked to form substituted or unsubstituted heterocyclic alkyl groups (e.g., 3 to 8, 3 to 6, 4 to 6, 4 to 5, or 5 to 6) or substituted or unsubstituted heteroaryl groups (e.g., 5 to 10, 5 to 9, or 5 to 6).

[0166] R 8 and R 10 They can be linked to form substituted or unsubstituted heterocyclic alkyl groups (e.g., 3 to 8, 3 to 6, 4 to 6, 4 to 5, or 5 to 6) or substituted or unsubstituted heteroaryl groups (e.g., 5 to 10, 5 to 9, or 5 to 6).

[0167] R 2A 、R 3A 、R 3B 、R 3C and R 5A and R AIndependently, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2 I, -OCH2F, substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4, or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2 to 8 members, 2 to 6 members, 4 to 6 members, 2 to 3 members, or 4 to 5 members), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6, or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8 members, 3 to 6 members, 4 to 6 members, 4 to 5 members, or 5 to 6 members), substituted or unsubstituted aryl (e.g., C6-C 10 or phenyl), or a substituted or unsubstituted heteroaryl (e.g., 5- to 10-membered, 5- to 9-membered, or 5- to 6-membered).

[0168] Those skilled in the art will understand that this compound can exist as a neutral species with counterions.

[0169] In the implementation scheme, the compound has the following formula:

[0170] B.L 1 、R 2 、R 3 and R 5 As described herein, it is included in the embodiments. A- is an anti-ion. In the embodiments, A - It is Cl - In an embodiment, A - It is Br - In an embodiment, A - It is H3CC(O)O - In an embodiment, A - It's F3CC(O)O - .

[0171] In an embodiment, the compound has the formula:

[0172] B.L 1 、R 2 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0173] In the implementation scheme, the compound has the following formula:

[0174] B.L 1 、R 2 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0175] In the implementation scheme, the compound has the following formula:

[0176] B.L 1 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0177] In the implementation scheme, the compound has the following formula:

[0178] B.L 1 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0179] In the embodiments, B is divalent cytosine or a derivative thereof, divalent guanine or a derivative thereof, divalent adenine or a derivative thereof, divalent thymine or a derivative thereof, divalent uracil or a derivative thereof, divalent hypoxanthine or a derivative thereof, divalent xanthine or a derivative thereof, divalent 7-methylguanine or a derivative thereof, divalent 5,6-dihydrouracil or a derivative thereof, divalent 5-methylcytosine or a derivative thereof, or divalent 5-hydroxymethylcytosine or a derivative thereof. In the embodiments, B is divalent cytosine or a derivative thereof, divalent guanine or a derivative thereof, divalent adenine or a derivative thereof, divalent thymine or a derivative thereof, or divalent uracil or a derivative thereof. In the embodiments, B is divalent cytosine or a derivative thereof. In the embodiments, B is divalent guanine or a derivative thereof. In the embodiments, B is divalent adenine or a derivative thereof. In the embodiments, B is divalent thymine or a derivative thereof. In the embodiments, B is divalent uracil or a derivative thereof.

[0180] In the implementation scheme, the compound has the following formula:

[0181] L 1 、R 2 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0182] In the implementation scheme, the compound has the following formula:

[0183] L1 、R 2 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0184] In an embodiment, the compound has the formula:

[0185] L 1 、R 2 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0186] In an embodiment, the compound has the formula:

[0187] L 1 、R 2 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0188] In an embodiment, the compound has the formula:

[0189] L 1 、R 2 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0190] In an embodiment, the compound has the formula:

[0191] L 1 、R 2 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0192] In an embodiment, the compound has the formula:

[0193] L 1 、R 2 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0194] In an embodiment, the compound has the formula:

[0195] L 1 、R 2 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0196] In the implementation scheme, the compound has the following formula:

[0197] L 1 、R 2 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0198] In the implementation scheme, the compound has the following formula:

[0199] L 1 、R 2 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0200] In the implementation scheme, the compound has the following formula:

[0201] L 1 、R 2 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0202] In the implementation scheme, the compound has the following formula:

[0203] L 1 、R 2 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0204] In the implementation scheme, the compound has the following formula:

[0205] L 1 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0206] In the implementation scheme, the compound has the following formula:

[0207] L 1 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0208] In the implementation scheme, the compound has the following formula:

[0209] L 1 、R3 and R 5 As described herein, it is included in the implementation scheme.

[0210] In the implementation scheme, the compound has the following formula:

[0211] L 1 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0212] In the implementation scheme, the compound has the following formula:

[0213] L 1 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0214] In the implementation scheme, the compound has the following formula:

[0215] L 1 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0216] In the implementation scheme, the compound has the following formula:

[0217] L 1 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0218] In the implementation scheme, the compound has the following formula:

[0219] L 1 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0220] In the implementation scheme, the compound has the following formula:

[0221] L 1 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0222] In the implementation scheme, the compound has the following formula:

[0223] L 1 、R 3 and R5 As described herein, it is included in the implementation scheme.

[0224] In an embodiment, the compound has the formula:

[0225] L 1 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0226] In an embodiment, the compound has the formula:

[0227] L 1 、R 3 and R 5 As described herein, it is included in the implementation scheme.

[0228] In an embodiment, L 1 It is a divalent linker containing 4 to 30 atoms.

[0229] In an embodiment, L 1 It's L 101 -L 102 -L 103 -L 104 -L 105 .

[0230] L 101 、L 102 、L 103 、L 104 and L 105 Independently, it is a bond, -NH-, -O-, -S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene (e.g., C1-C8, C1-C6, C1-C4, or C1-C2), substituted or unsubstituted heteroalkylene (e.g., 2 to 8-membered, 2 to 6-membered, 4 to 6-membered, 2 to 3-membered, or 4 to 5-membered), substituted or unsubstituted cycloalkylene (e.g., C3-C8, C3-C6, C4-C6, or C5-C6), substituted or unsubstituted heteroalkylene (e.g., 3 to 8-membered, 3 to 6-membered, 4 to 6-membered, 4 to 5-membered, or 5 to 6-membered), substituted or unsubstituted arylene (e.g., C6-C 10 or phenylene), or a substituted or unsubstituted heteroarylene (e.g., 5- to 10-membered, 5- to 9-membered, or 5- to 6-membered).

[0231] In an embodiment, substituted L 101(e.g., substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene and / or substituted heteroarylene) is substituted with at least one substituent, size-restricted substituent, or lower substituent; wherein if the substituted L 101 is substituted with a plurality of groups selected from substituents, size-limited substituents and lower substituents; each substituent, size-limited substituent and / or lower substituent may optionally be different. In an embodiment, when L 101 When substituted, it is replaced by at least one substituent. In the embodiment, when L 101 When substituted, it is replaced by at least one size-restricted substituent. In the embodiment, when L 101 When substituted, it is substituted with at least one lower substituent.

[0232] In an embodiment, L 101 It is a key. In the implementation plan, L 101 is -NH-. In an embodiment, L 101 Yes -O-. In the implementation plan, L 101 Yes -S-. In the implementation plan, L 101 is -S(O)-. In an embodiment, L 101 It is -S(O)2-. In the implementation scheme, L 101 is -C(O)-. In an embodiment, L 101 It is -C(O)NH-. In the implementation scheme, L 101 is -NHC(O)-. In an embodiment, L 101 is -NHC(O)NH-. In an embodiment, L 101 is -C(O)O-. In an embodiment, L 101 is -OC(O)-. In an embodiment, L 101 It is a substituted or unsubstituted C1-C4 alkylene group. In the embodiments, L 101 is a substituted or unsubstituted 2- to 6-membered heteroalkylene group.

[0233] In an embodiment, substituted L 102 (e.g., substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene and / or substituted heteroarylene) is substituted with at least one substituent, size-restricted substituent, or lower substituent; wherein if the substituted L 102 is substituted with a plurality of groups selected from substituents, size-limited substituents and lower substituents; each substituent, size-limited substituent and / or lower substituent may optionally be different. In an embodiment, when L 102 When substituted, it is replaced by at least one substituent. In the embodiment, when L 102When substituted, it is replaced by at least one size-restricted substituent. In the embodiment, when L 102 When substituted, it is substituted with at least one lower substituent.

[0234] In an embodiment, L 102 It is a key. In the implementation plan, L 102 is -NH-. In an embodiment, L 102 Yes -O-. In the implementation plan, L 102 Yes -S-. In the implementation plan, L 102 is -S(O)-. In an embodiment, L 102 It is -S(O)2-. In the implementation scheme, L 102 is -C(O)-. In an embodiment, L 102 It is -C(O)NH-. In the implementation scheme, L 102 is -NHC(O)-. In an embodiment, L 102 is -NHC(O)NH-. In an embodiment, L 102 is -C(O)O-. In an embodiment, L 102 is -OC(O)-. In an embodiment, L 102 It is a substituted or unsubstituted C1-C4 alkylene group. In the embodiments, L 102 is a substituted or unsubstituted 2 to 6 membered heteroalkylene group. 102 is an unsubstituted 3 to 8 membered heterocycloalkyl. 102 is unsubstituted piperidinyl. In an embodiment, L 102 yes

[0235] In an embodiment, substituted L 103 (e.g., substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene and / or substituted heteroarylene) is substituted with at least one substituent, size-restricted substituent, or lower substituent; wherein if the substituted L 103 is substituted with a plurality of groups selected from substituents, size-limited substituents and lower substituents; each substituent, size-limited substituent and / or lower substituent may optionally be different. In an embodiment, when L 103 When substituted, it is replaced by at least one substituent. In the embodiment, when L 103 When substituted, it is replaced by at least one size-restricted substituent. In the embodiment, when L 103 When substituted, it is substituted with at least one lower substituent.

[0236] In an embodiment, L 103It is a key. In the implementation plan, L 103 is -NH-. In an embodiment, L 103 Yes -O-. In the implementation plan, L 103 Yes -S-. In the implementation plan, L 103 is -S(O)-. In an embodiment, L 103 It is -S(O)2-. In the implementation scheme, L 103 is -C(O)-. In an embodiment, L 103 It is -C(O)NH-. In the implementation scheme, L 103 is -NHC(O)-. In an embodiment, L 103 is -NHC(O)NH-. In an embodiment, L 103 is -C(O)O-. In an embodiment, L 103 is -OC(O)-. In an embodiment, L 103 It is a substituted or unsubstituted C1-C4 alkylene group. In the embodiments, L 103 is a substituted or unsubstituted 2- to 6-membered heteroalkylene group.

[0237] In an embodiment, substituted L 104 (e.g., substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene and / or substituted heteroarylene) is substituted with at least one substituent, size-restricted substituent, or lower substituent; wherein if the substituted L 104 is substituted with a plurality of groups selected from substituents, size-limited substituents and lower substituents; each substituent, size-limited substituent and / or lower substituent may optionally be different. In an embodiment, when L 104 When substituted, it is replaced by at least one substituent. In the embodiment, when L 104 When substituted, it is replaced by at least one size-restricted substituent. In the embodiment, when L 104 When substituted, it is substituted with at least one lower substituent.

[0238] In an embodiment, L 104 It is a key. In the implementation plan, L 104 is -NH-. In an embodiment, L 104 Yes -O-. In the implementation plan, L 104 Yes -S-. In the implementation plan, L 104 is -S(O)-. In an embodiment, L 104 It is -S(O)2-. In the implementation scheme, L 104 is -C(O)-. In an embodiment, L 104 It is -C(O)NH-. In the implementation scheme, L 104is -NHC(O)-. In an embodiment, L 104 is -NHC(O)NH-. In an embodiment, L 104 is -C(O)O-. In an embodiment, L 104 is -OC(O)-. In an embodiment, L 104 is an unsubstituted C1-C 10 In an embodiment, L 104 is an unsubstituted methylene group. In an embodiment, L 104 is unsubstituted ethylene. In an embodiment, L 104 is unsubstituted propylene. In an embodiment, L 104 is unsubstituted n-propylene. In an embodiment, L 104 is unsubstituted butylene. In an embodiment, L 104 is unsubstituted n-butylene. In an embodiment, L 104 It is an unsubstituted pentylene. In the implementation scheme, L 104 is unsubstituted n-pentylene. In an embodiment, L 104 It is an unsubstituted hexyl group. In the implementation plan, L 104 is unsubstituted n-hexylene. In an embodiment, L 104 It is an unsubstituted heptyl group. In the implementation scheme, L 104 is unsubstituted n-heptylene. In an embodiment, L 104 It is unsubstituted acetone. In the implementation plan, L 104 is unsubstituted n-octylene. In an embodiment, L 104 It is an unsubstituted C2-C6 ynylene group. In the implementation scheme, L 104 It is an unsubstituted ethynyl group. In the implementation scheme, L 104 It is an unsubstituted propyne group. In the implementation scheme, L 104 is unsubstituted butynylene. In an embodiment, L 104 is an unsubstituted pentynylene. In an embodiment, L 104 It is an unsubstituted hexyneylyl group. In the implementation scheme, L 104 yes In an embodiment, L 104 is a substituted or unsubstituted 2 to 6 membered heteroalkylene group. 104 It is an unsubstituted 2- to 6-membered heteroalkylene group. In the embodiments, L 104 yes In an embodiment, L 104 yes wherein n104 is an integer from 1 to 10. In an embodiment, n104 is 1. In an embodiment, n104 is 2. In an embodiment, n104 is 3. In an embodiment, n104 is 4. In an embodiment, n104 is 5. In an embodiment, n104 is 6. In an embodiment, n104 is 7. In an embodiment, n104 is 8. In an embodiment, n104 is 9. In an embodiment, n104 is 10. In an embodiment, L 104 is a substituted or unsubstituted phenylene group. In an embodiment, L 104 It is an unsubstituted phenylene. In the implementation scheme, L 104 yes

[0239] In an embodiment, substituted L 105 (e.g., substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene and / or substituted heteroarylene) is substituted with at least one substituent, size-restricted substituent, or lower substituent; wherein if the substituted L 105 is substituted with a plurality of groups selected from substituents, size-limited substituents and lower substituents; each substituent, size-limited substituent and / or lower substituent may optionally be different. In an embodiment, when L 105 When substituted, it is substituted with at least one substituent. In an embodiment, when L 105 When substituted, it is replaced by at least one size-restricted substituent. In the embodiment, when L 105 When substituted, it is substituted with at least one lower substituent.

[0240] In an embodiment, L 105 is a bond. In an embodiment, L 105 is -NH-. In an embodiment, L 105 is -O-. In an embodiment, L 105 is -S-. In an embodiment, L 105 is -S(O)-. In an embodiment, L 105 is -S(O)2-. In an embodiment, L 105 is -C(O)-. In an embodiment, L 105 is -C(O)NH-. In an embodiment, L 105 is -NHC(O)-. In an embodiment, L 105 is -NHC(O)NH-. In an embodiment, L 105 is -C(O)O-. In an embodiment, L 105 It is -OC(O)-. In the implementation scheme, L 105 is an unsubstituted C1-C 10In an embodiment, L 105 It is a substituted or unsubstituted C1-C4 alkylene group. In the embodiments, L 105 is an unsubstituted methylene group. In an embodiment, L 105 is unsubstituted ethylene. In an embodiment, L 105 is unsubstituted propylene. In an embodiment, L 105 is unsubstituted n-propylene. In an embodiment, L 105 is unsubstituted butylene. In an embodiment, L 105 is unsubstituted n-butylene. In an embodiment, L 105 It is an unsubstituted pentylene. In the implementation scheme, L 105 is unsubstituted n-pentylene. In an embodiment, L 105 It is an unsubstituted hexyl group. In the implementation plan, L 105 is unsubstituted n-hexylene. In an embodiment, L 105 It is an unsubstituted heptyl group. In the implementation scheme, L 105 is unsubstituted n-heptylene. In an embodiment, L 105 It is unsubstituted acetone. In the implementation plan, L 105 is unsubstituted n-octylene. In an embodiment, L 105 It is an unsubstituted C2-C6 ynylene group. In the implementation scheme, L 105 It is an unsubstituted ethynyl group. In the implementation scheme, L 105 It is an unsubstituted propyne group. In the implementation scheme, L 105 is unsubstituted butynylene. In an embodiment, L 105 is an unsubstituted pentynylene. In an embodiment, L 105 It is an unsubstituted hexyneylyl group. In the implementation scheme, L 105 yes In an embodiment, L 105 It is a substituted 2 to 8-membered heteroalkylene group. In the embodiments, L 105 It is an oxo-substituted 2- to 8-membered heteroalkylene group. In the embodiments, L 105 is an oxo-substituted 2- to 8-membered heteroalkenylene. In an embodiment, L 105 yes In an embodiment, L 105 yes In an embodiment, L 105 yes In an embodiment, L 105 is a substituted or unsubstituted 2 to 8 membered heteroalkynylene. 105 yes In an embodiment, L 105 yes wherein n105 is an integer from 1 to 10. In an embodiment, n105 is 1. In an embodiment, n105 is 2. In an embodiment, n105 is 3. In an embodiment, n105 is 4. In an embodiment, n105 is 5. In an embodiment, n105 is 6. In an embodiment, n105 is 7. In an embodiment, n105 is 8. In an embodiment, n105 is 9. In an embodiment, n105 is 10. In an embodiment, L 105 It is an unsubstituted 5- to 10-membered heteroaryl group. In the implementation scheme, L 105 is an unsubstituted triazolylene. In an embodiment, L 105 yes

[0241] In an embodiment, L 1 yes In an embodiment, L 1 yes In an embodiment, L 1 yes In an embodiment, L 1 yes In an embodiment, L 1 yes In an embodiment, L 1 yes In an embodiment, L 1 yes In an embodiment, L 1 yes In an embodiment, L 1 yes In an embodiment, L 1 yes In an embodiment, L 1 yes

[0242] In an embodiment, substituted R 1 (e.g., substituted alkyl and / or substituted heteroalkyl) is substituted by at least one substituent, a size-restricted substituent, or a lower substituent; wherein if the substituted R 1 is substituted with a plurality of groups selected from substituents, size-limited substituents and lower substituents; each substituent, size-limited substituent and / or lower substituent may optionally be different. In an embodiment, when R 1 When substituted, it is replaced by at least one substituent. In the implementation, when R 1 When substituted, it is replaced by at least one size-restricted substituent. In the embodiment, when R 1 When substituted, it is substituted with at least one lower substituent.

[0243] In an embodiment, R 1 is hydrogen, CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4, or C1-C2), or substituted or unsubstituted heteroalkyl (e.g., 2-8, 2-6, 4-6, 2-3, or 4-5 members).

[0244] In an embodiment, R 1 is hydrogen. In an embodiment, R 1 It is an unsubstituted C1-C4 alkyl group. In the embodiments, R 1 is unsubstituted methyl. In an embodiment, R 1 is an unsubstituted ethyl group. In an embodiment, R 1 is unsubstituted propyl. In an embodiment, R 1 It is an unsubstituted n-propyl group. In the implementation scheme, R 1 is unsubstituted isopropyl. In an embodiment, R 1 is unsubstituted butyl. In an embodiment, R 1 is unsubstituted n-butyl. In an embodiment, R 1 is unsubstituted isobutyl. In an embodiment, R 1 is an unsubstituted tert-butyl group.

[0245] In an embodiment, R 2 is hydrogen or -OH. In an embodiment, R 2 is hydrogen. In an embodiment, R 2 Yes-OR 2A In an embodiment, R 2 It is -OH.

[0246] In an embodiment, substituted R 2A (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, and / or substituted heteroaryl) is substituted with at least one substituent, size-restricted substituent, or lower substituent; wherein if the substituted R 2A is substituted with a plurality of groups selected from substituents, size-limited substituents and lower substituents; each substituent, size-limited substituent and / or lower substituent may optionally be different. In an embodiment, when R 2A When substituted, it is replaced by at least one substituent. In the implementation, when R 2AWhen substituted, it is substituted with at least one substituent of limited size. In an embodiment, when R 2A When substituted, it is substituted with at least one lower substituent.

[0247] In an embodiment, R 2A It is hydrogen. In the implementation scheme, R 2A It is an unsubstituted C1-C4 alkyl group. In the embodiments, R 2A is unsubstituted methyl. In an embodiment, R 2A is an unsubstituted ethyl group. In an embodiment, R 2A is unsubstituted propyl. In an embodiment, R 2A is unsubstituted n-propyl. In an embodiment, R 2A It is an unsubstituted isopropyl group. In the implementation scheme, R 2A It is an unsubstituted butyl. In the implementation scheme, R 2A It is an unsubstituted n-butyl. In the implementation scheme, R 2A It is an unsubstituted isobutyl group. In the implementation scheme, R 2A It is unsubstituted tert-butyl.

[0248] In an embodiment, R 3 Yes - OR 3A In an embodiment, R 3 is -OH. In an embodiment, R 3 Yes -OP(NR) 3B R 3C )-OR 3A In an embodiment, R 3 yes

[0249] In an embodiment, substituted R 3A (For example, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl and / or substituted heteroaryl) is substituted by at least one substituent, a size-restricted substituent, or a lower substituent; wherein if the substituted R 3A The substituent is replaced by a plurality of groups selected from substituents, size-restricted substituents, and lower substituents; each substituent, size-restricted substituent, and / or lower substituent may optionally be different. In an embodiment, when R 3A When substituted, it is replaced by at least one substituent. In the implementation, when R 3A When substituted, it is substituted with at least one substituent of limited size. In an embodiment, when R 3A When substituted, it is substituted with at least one lower substituent.

[0250] In an embodiment, R 3A It is hydrogen. In the implementation scheme, R3A It is an unsubstituted C1-C4 alkyl group. In the embodiments, R 3A is unsubstituted methyl. In an embodiment, R 3A is an unsubstituted ethyl group. In an embodiment, R 3A is unsubstituted propyl. In an embodiment, R 3A It is an unsubstituted n-propyl group. In the implementation scheme, R 3A is unsubstituted isopropyl. In an embodiment, R 3A is unsubstituted butyl. In an embodiment, R 3A is unsubstituted n-butyl. In an embodiment, R 3A is unsubstituted isobutyl. In an embodiment, R 3A is unsubstituted tert-butyl. In an embodiment, R 3A is a substituted C1-C4 alkyl. In an embodiment, R 3A is a cyano-substituted C1-C4 alkyl group. 3A yes

[0251] In an embodiment, substituted R 3B (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, and / or substituted heteroaryl) is substituted with at least one substituent, size-restricted substituent, or lower substituent; wherein if the substituted R 3B is substituted with a plurality of groups selected from substituents, size-limited substituents and lower substituents; each substituent, size-limited substituent and / or lower substituent may optionally be different. In an embodiment, when R 3B When substituted, it is replaced by at least one substituent. In the implementation, when R 3B When substituted, it is replaced by at least one size-restricted substituent. In the embodiment, when R 3B When substituted, it is substituted with at least one lower substituent.

[0252] In an embodiment, R 3B is hydrogen. In an embodiment, R 3B It is an unsubstituted C1-C4 alkyl group. In the embodiments, R 3B is unsubstituted methyl. In an embodiment, R 3B is an unsubstituted ethyl group. In an embodiment, R 3B is unsubstituted propyl. In an embodiment, R 3B It is an unsubstituted n-propyl group. In the implementation scheme, R 3B is unsubstituted isopropyl. In an embodiment, R 3B is unsubstituted butyl. In an embodiment, R 3Bis unsubstituted n-butyl. In an embodiment, R 3B is unsubstituted isobutyl. In an embodiment, R 3B is an unsubstituted tert-butyl group.

[0253] In an embodiment, substituted R 3C (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, and / or substituted heteroaryl) is substituted with at least one substituent, size-restricted substituent, or lower substituent; wherein if the substituted R 3C is substituted with a plurality of groups selected from substituents, size-limited substituents and lower substituents; each substituent, size-limited substituent and / or lower substituent may optionally be different. In an embodiment, when R 3C When substituted, it is replaced by at least one substituent. In the implementation, when R 3C When substituted, it is replaced by at least one size-restricted substituent. In the embodiment, when R 3C When substituted, it is substituted with at least one lower substituent.

[0254] In an embodiment, R 3C is hydrogen. In an embodiment, R 3C It is an unsubstituted C1-C4 alkyl group. In the embodiments, R 3C is unsubstituted methyl. In an embodiment, R 3C is an unsubstituted ethyl group. In an embodiment, R 3C is unsubstituted propyl. In an embodiment, R 3C It is an unsubstituted n-propyl group. In the implementation scheme, R 3C is unsubstituted isopropyl. In an embodiment, R 3C is unsubstituted butyl. In an embodiment, R 3C is unsubstituted n-butyl. In an embodiment, R 3C is unsubstituted isobutyl. In an embodiment, R 3C is an unsubstituted tert-butyl group.

[0255] In an embodiment, R 4 is hydrogen. In an embodiment, R 4 It is an unsubstituted methyl group.

[0256] In the implementation plan, when R 2 and R 4 The substituted ring formed when the substituents are linked (e.g., a substituted heterocycloalkyl) is substituted with at least one substituent, a size-restricted substituent, or a lower substituent; wherein when R 2 and R 4When substituents are linked, the resulting substituent ring is replaced by a plurality of groups selected from substituents, size-restricted substituents, and lower substituents; each substituent, size-restricted substituent, and / or lower substituent may optionally be different. In an embodiment, when R 2 and R 4 When the substituted ring formed when the substituents are attached is substituted, it is substituted with at least one substituent. 2 and R 4 When a substituent ring formed during substituent linkage is substituted, it is substituted by at least one size-constrained substituent. In an embodiment, when R 2 and R 4 When a substituted ring formed when substituents are linked is substituted, it is substituted with at least one lower substituent.

[0257] In an embodiment, R 2 and R 4 Substituents connect to form substituted or unsubstituted 3- to 8-membered heterocyclic alkyl groups. In the embodiments, R 2 and R 4 Substituents are linked to form substituted or unsubstituted tetrahydrofuran groups.

[0258] In an embodiment, R 5 It is -OH.

[0259] In an embodiment, substituted R 5A (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, and / or substituted heteroaryl) is substituted with at least one substituent, size-restricted substituent, or lower substituent; wherein if the substituted R 5A is substituted with a plurality of groups selected from substituents, size-limited substituents and lower substituents; each substituent, size-limited substituent and / or lower substituent may optionally be different. In an embodiment, when R 5A When substituted, it is replaced by at least one substituent. In the implementation, when R 5A When substituted, it is replaced by at least one size-restricted substituent. In the embodiment, when R 5A When substituted, it is substituted with at least one lower substituent.

[0260] In an embodiment, R 5A It is hydrogen or a substituted C1-C6 alkyl group. In the embodiments, R 5A is hydrogen. In an embodiment, R 5A It is an unsubstituted C1-C4 alkyl group. In the embodiments, R 5A is unsubstituted methyl. In an embodiment, R 5A is an unsubstituted ethyl group. In an embodiment, R 5Ais unsubstituted propyl. In an embodiment, R 5A is unsubstituted n-propyl. In an embodiment, R 5A It is an unsubstituted isopropyl group. In the implementation scheme, R 5A It is an unsubstituted butyl. In the implementation scheme, R 5A It is an unsubstituted n-butyl. In the implementation scheme, R 5A It is an unsubstituted isobutyl group. In the implementation scheme, R SA is unsubstituted tert-butyl. In an embodiment, R SA is a substituted C1-C6 alkyl. In an embodiment, R 5A It is dimethoxytriphenylmethyl. In the implementation scheme, R 5A yes

[0261] In an embodiment, substituted R 6 (For example, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl and / or substituted heteroaryl) is substituted by at least one substituent, a size-restricted substituent, or a lower substituent; wherein if the substituted R 6 The substituent is replaced by a plurality of groups selected from substituents, size-restricted substituents, and lower substituents; each substituent, size-restricted substituent, and / or lower substituent may optionally be different. In an embodiment, when R 6 When substituted, it is replaced by at least one substituent. In the implementation, when R 6 When substituted, it is substituted with at least one substituent of limited size. In an embodiment, when R 6 When substituted, it is substituted with at least one lower substituent.

[0262] In an embodiment, R 6 It is hydrogen. In the implementation scheme, R 6 It is a halogen. In the implementation plan, R 6 is -F. In an embodiment, R 6 Yes -Cl. In the implementation plan, R 6 is -Br. In an embodiment, R 6 In an embodiment, R 6 is -CCl3. In an embodiment, R 6 is -CBr3. In an embodiment, R 6 Yes, it is -CF3. In the implementation plan, R 6 In an embodiment, R 6 is -CH2Cl. In an embodiment, R 6 is -CH2Br. In an embodiment, R 6 It is -CH2F. In the implementation scheme, R6 is -CH2I. In an embodiment, R 6 is -CHCl2. In an embodiment, R 6 is -CHBr2. In an embodiment, R 6 is -CHF2. In an embodiment, R 6 is -CHI2. In an embodiment, R 6 is -CN. In an embodiment, R 6 is -OH. In an embodiment, R 6 It is -NH2. In the implementation plan, R 6 is -COOH. In an embodiment, R 6 Yes -CONH2. In the implementation plan, R 6 is -NO2. In an embodiment, R 6 In an embodiment, R 6 Yes-SO3R A In an embodiment, R 6 is -SO3H. In an embodiment, R 6 is -SO2NH2. In an embodiment, R 6 It is -NHNH2. In the implementation plan, R 6 is -ONH2. In an embodiment, R 6 It is -NHC(O)NH2. In the implementation scheme, R 6 is -NHSO2H. In an embodiment, R 6 is -NHC(O)H. In an embodiment, R 6 is -NHC(O)OH. In an embodiment, R 6 is -NHOH. In an embodiment, R 6 is -OCCl3. In an embodiment, R 6 is -OCBr3. In an embodiment, R 6 is -OCF3. In an embodiment, R 6 Yes -OCI3. In the implementation scheme, R 6 is -OCH2Cl. In an embodiment, R 6 is -OCH2Br. In an embodiment, R 6 is -OCH2F. In an embodiment, R 6 is -OCH2I. In an embodiment, R 6 It is -OCHCl2. In the implementation scheme, R 6 is -OCHBr2. In an embodiment, R 6 is -OCHF2. In an embodiment, R 6 In one embodiment, R 6In an embodiment, R 6 In an embodiment, R 6 It is an unsubstituted C1-C4 alkyl group. In the embodiments, R 6 is unsubstituted methyl. In an embodiment, R 6 is an unsubstituted ethyl group. In an embodiment, R 6 is unsubstituted propyl. In an embodiment, R 6 is unsubstituted n-propyl. In an embodiment, R 6 It is an unsubstituted isopropyl group. In the implementation scheme, R 6 It is an unsubstituted butyl. In the implementation scheme, R 6 It is an unsubstituted n-butyl. In the implementation scheme, R 6 It is an unsubstituted isobutyl group. In the implementation scheme, R 6 is unsubstituted tert-butyl. In an embodiment, R 6 It is an unsubstituted 2- to 6-membered heteroalkyl group. In the embodiments, R 6 It is an unsubstituted methoxy group. In the embodiments, R 6 It is an unsubstituted ethoxy group. In the embodiments, R 6 is unsubstituted propoxy. In an embodiment, R 6 is unsubstituted n-propoxy. In an embodiment, R 6 is unsubstituted isopropoxy. In an embodiment, R 6 It is an unsubstituted butoxy group.

[0263] In an embodiment, substituted R 7 (For example, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl and / or substituted heteroaryl) is substituted by at least one substituent, a size-restricted substituent, or a lower substituent; wherein if the substituted R 7 The substituent is replaced by a plurality of groups selected from substituents, size-restricted substituents, and lower substituents; each substituent, size-restricted substituent, and / or lower substituent may optionally be different. In an embodiment, when R 7 When substituted, it is replaced by at least one substituent. In the implementation, when R 7 When substituted, it is substituted with at least one substituent of limited size. In an embodiment, when R 7 When substituted, it is substituted with at least one lower substituent.

[0264] In an embodiment, R 7 It is hydrogen. In the implementation scheme, R 7 It is a halogen. In the implementation plan, R 7 is -F. In an embodiment, R 7Yes -Cl. In the implementation plan, R 7 is -Br. In an embodiment, R 7 In an embodiment, R 7 is -CCl3. In an embodiment, R 7 is -CBr3. In an embodiment, R 7 Yes, it is -CF3. In the implementation plan, R 7 In an embodiment, R 7 is -CH2Cl. In an embodiment, R 7 is -CH2Br. In an embodiment, R 7 It is -CH2F. In the implementation scheme, R 7 is -CH2I. In an embodiment, R 7 is -CHCl2. In an embodiment, R 7 is -CHBr2. In an embodiment, R 7 is -CHF2. In an embodiment, R 7 is -CHI2. In an embodiment, R 7 is -CN. In an embodiment, R 7 is -OH. In an embodiment, R 7 It is -NH2. In the implementation plan, R 7 is -COOH. In an embodiment, R 7 Yes -CONH2. In the implementation plan, R 7 is -NO2. In an embodiment, R 7 In an embodiment, R 7 Yes-SO3R A In an embodiment, R 7 is -SO3H. In an embodiment, R 7 is -SO2NH2. In an embodiment, R 7 It is -NHNH2. In the implementation plan, R 7 is -ONH2. In an embodiment, R 7 It is -NHC(O)NH2. In the implementation scheme, R 7 is -NHSO2H. In an embodiment, R 7 is -NHC(O)H. In an embodiment, R 7 is -NHC(O)OH. In an embodiment, R 7 is -NHOH. In an embodiment, R 7 is -OCCl3. In an embodiment, R 7 is -OCBr3. In an embodiment, R 7 is -OCF3. In an embodiment, R 7In an embodiment, R 7 It is -OCH2Cl. In the implementation scheme, R 7 is -OCH2Br. In an embodiment, R 7 is -OCH2F. In an embodiment, R 7 It is -OCH2I. In the implementation scheme, R 7 It is -OCHCl2. In the implementation scheme, R 7 is -OCHBr2. In an embodiment, R 7 is -OCHF2. In an embodiment, R 7 In one embodiment, R 7 In an embodiment, R 7 It is -N3. In the implementation plan, R 7 It is an unsubstituted C1-C4 alkyl group. In the embodiments, R 7 is unsubstituted methyl. In an embodiment, R 7 is an unsubstituted ethyl group. In an embodiment, R 7 is unsubstituted propyl. In an embodiment, R 7 It is an unsubstituted n-propyl group. In the implementation scheme, R 7 is unsubstituted isopropyl. In an embodiment, R 7 is unsubstituted butyl. In an embodiment, R 7 is unsubstituted n-butyl. In an embodiment, R 7 is unsubstituted isobutyl. In an embodiment, R 7 is unsubstituted tert-butyl. In an embodiment, R 7 It is an unsubstituted 2- to 6-membered heteroalkyl group. In the embodiments, R 7 It is an unsubstituted methoxy group. In the embodiments, R 7 It is an unsubstituted ethoxy group. In the embodiments, R 7 It is an unsubstituted propoxy group. In the embodiments, R 7 It is an unsubstituted propoxy group. In the embodiments, R 7 It is an unsubstituted isopropoxy group. In the embodiments, R 7 It is an unsubstituted butoxy group.

[0265] In an embodiment, substituted R 8 (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, and / or substituted heteroaryl) is substituted with at least one substituent, size-restricted substituent, or lower substituent; wherein if the substituted R 8The substituent is replaced by a plurality of groups selected from substituents, size-restricted substituents, and lower substituents; each substituent, size-restricted substituent, and / or lower substituent may optionally be different. In an embodiment, when R 8 When substituted, it is replaced by at least one substituent. In the implementation, when R 8 When substituted, it is substituted with at least one substituent of limited size. In an embodiment, when R 8 When substituted, it is substituted with at least one lower substituent.

[0266] In an embodiment, R 8 It is hydrogen. In the implementation scheme, R 8 It is a halogen. In the implementation plan, R 8 is -F. In an embodiment, R 8 Yes -Cl. In the implementation plan, R 8 is -Br. In an embodiment, R 8 In an embodiment, R 8 is -CCl3. In an embodiment, R 8 is -CBr3. In an embodiment, R 8 Yes, it is -CF3. In the implementation plan, R 8 In an embodiment, R 8 is -CH2Cl. In an embodiment, R 8 is -CH2Br. In an embodiment, R 8 It is -CH2F. In the implementation scheme, R 8 is -CH2I. In an embodiment, R 8 is -CHCl2. In an embodiment, R 8 is -CHBr2. In an embodiment, R 8 is -CHF2. In an embodiment, R 8 is -CHI2. In an embodiment, R 8 is -CN. In an embodiment, R 8 is -OH. In an embodiment, R 8 It is -NH2. In the implementation plan, R 8 is -COOH. In an embodiment, R 8 Yes -CONH2. In the implementation plan, R 8 is -NO2. In an embodiment, R 8 In an embodiment, R 8 Yes-SO3R A In an embodiment, R 8 is -SO3H. In an embodiment, R 8 is -SO2NH2. In an embodiment, R8 It is -NHNH2. In the implementation plan, R 8 Yes -ONH2. In the implementation scheme, R 8 It is -NHC(O)NH2. In the implementation scheme, R 8 It is -NHSO2H. In the implementation plan, R 8 It is -NHC(O)H. In the implementation scheme, R 8 It is -NHC(O)OH. In the implementation scheme, R 8 It is -NHOH. In the implementation scheme, R 8 It is -OCCl3. In the implementation scheme, R 8 It is -OCBr3. In the implementation scheme, R 8 Yes -OCF3. In the implementation scheme, R 8 In an embodiment, R 8 It is -OCH2Cl. In the implementation scheme, R 8 is -OCH2Br. In an embodiment, R 8 is -OCH2F. In an embodiment, R 8 It is -OCH2I. In the implementation scheme, R 8 It is -OCHCl2. In the implementation scheme, R 8 is -OCHBr2. In an embodiment, R 8 is -OCHF2. In an embodiment, R 8 In one embodiment, R 8 In an embodiment, R 8 It is -N3. In the implementation plan, R 8 It is an unsubstituted C1-C4 alkyl group. In the embodiments, R 8 is unsubstituted methyl. In an embodiment, R 8 is an unsubstituted ethyl group. In an embodiment, R 8 is unsubstituted propyl. In an embodiment, R 8 It is an unsubstituted n-propyl group. In the implementation scheme, R 8 is unsubstituted isopropyl. In an embodiment, R 8 is unsubstituted butyl. In an embodiment, R 8 is unsubstituted n-butyl. In an embodiment, R 8 is unsubstituted isobutyl. In an embodiment, R 8 is unsubstituted tert-butyl. In an embodiment, R 8 It is an unsubstituted 2- to 6-membered heteroalkyl group. In the embodiments, R 8 It is an unsubstituted methoxy group. In the embodiments, R 8It is an unsubstituted ethoxy group. In the embodiments, R 8 is unsubstituted propoxy. In an embodiment, R 8 is unsubstituted n-propoxy. In an embodiment, R 8 is unsubstituted isopropoxy. In an embodiment, R 8 It is an unsubstituted butoxy group.

[0267] In an embodiment, substituted R 9 (For example, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl and / or substituted heteroaryl) is substituted by at least one substituent, a size-restricted substituent, or a lower substituent; wherein if the substituted R 9 The substituent is replaced by a plurality of groups selected from substituents, size-restricted substituents, and lower substituents; each substituent, size-restricted substituent, and / or lower substituent may optionally be different. In an embodiment, when R 9 When substituted, it is replaced by at least one substituent. In the implementation, when R 9 When substituted, it is substituted with at least one substituent of limited size. In an embodiment, when R 9 When substituted, it is substituted with at least one lower substituent.

[0268] In an embodiment, R 9 It is hydrogen. In the implementation scheme, R 9 It is a halogen. In the implementation plan, R 9 is -F. In an embodiment, R 9 Yes -Cl. In the implementation plan, R 9 is -Br. In an embodiment, R 9 In an embodiment, R 9 is -CCl3. In an embodiment, R 9 is -CBr3. In an embodiment, R 9 Yes, it is -CF3. In the implementation plan, R 9 In an embodiment, R 9 is -CH2Cl. In an embodiment, R 9 is -CH2Br. In an embodiment, R 9 It is -CH2F. In the implementation scheme, R 9 is -CH2I. In an embodiment, R 9 is -CHCl2. In an embodiment, R 9 is -CHBr2. In an embodiment, R 9 is -CHF2. In an embodiment, R 9 is -CHI2. In an embodiment, R 9Yes -CN. In the implementation plan, R 9 It is -OH. In the implementation scheme, R 9 is -NH2. In an embodiment, R 9 It is -COOH. In the implementation scheme, R 9 Yes -CONH2. In the implementation plan, R 9 It is -NO2. In the implementation plan, R 9 Yes -SH. In the implementation plan, R 9 Yes - SO3R A In an embodiment, R 9 is -SO3H. In an embodiment, R 9 is -SO2NH2. In an embodiment, R 9 It is -NHNH2. In the implementation plan, R 9 Yes -ONH2. In the implementation scheme, R 9 It is -NHC(O)NH2. In the implementation scheme, R 9 It is -NHSO2H. In the implementation plan, R 9 It is -NHC(O)H. In the implementation scheme, R 9 It is -NHC(O)OH. In the implementation scheme, R 9 It is -NHOH. In the implementation scheme, R 9 It is -OCCl3. In the implementation scheme, R 9 It is -OCBr3. In the implementation scheme, R 9 Yes -OCF3. In the implementation scheme, R 9 In an embodiment, R 9 It is -OCH2Cl. In the implementation scheme, R 9 is -OCH2Br. In an embodiment, R 9 is -OCH2F. In an embodiment, R 9 It is -OCH2I. In the implementation scheme, R 9 It is -OCHCl2. In the implementation scheme, R 9 is -OCHBr2. In an embodiment, R 9 is -OCHF2. In an embodiment, R 9 In one embodiment, R 9 In an embodiment, R 9 It is -N3. In the implementation plan, R 9 It is an unsubstituted C1-C4 alkyl group. In the embodiments, R 9 is unsubstituted methyl. In an embodiment, R 9 is an unsubstituted ethyl group. In an embodiment, R 9is unsubstituted propyl. In an embodiment, R 9 is unsubstituted n-propyl. In an embodiment, R 9 It is an unsubstituted isopropyl group. In the implementation scheme, R 9 It is an unsubstituted butyl. In the implementation scheme, R 9 It is an unsubstituted n-butyl. In the implementation scheme, R 9 It is an unsubstituted isobutyl group. In the implementation scheme, R 9 is unsubstituted tert-butyl. In an embodiment, R 9 It is an unsubstituted 2- to 6-membered heteroalkyl group. In the embodiments, R 9 It is an unsubstituted methoxy group. In the embodiments, R 9 It is an unsubstituted ethoxy group. In the embodiments, R 9 is unsubstituted propoxy. In an embodiment, R 9 is unsubstituted n-propoxy. In an embodiment, R 9 is unsubstituted isopropoxy. In an embodiment, R 9 It is an unsubstituted butoxy group.

[0269] In an embodiment, substituted R A (For example, substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl and / or substituted heteroaryl) is substituted by at least one substituent, a size-restricted substituent, or a lower substituent; wherein if the substituted R A The substituent is replaced by a plurality of groups selected from substituents, size-restricted substituents, and lower substituents; each substituent, size-restricted substituent, and / or lower substituent may optionally be different. In an embodiment, when R A When substituted, it is replaced by at least one substituent. In the implementation, when R A When substituted, it is substituted with at least one substituent of limited size. In an embodiment, when R A When substituted, it is substituted with at least one lower substituent.

[0270] In an embodiment, R A It is hydrogen. In the implementation scheme, R A It is an unsubstituted C1-C4 alkyl group. In the embodiments, R A is unsubstituted methyl. In an embodiment, R A is an unsubstituted ethyl group. In an embodiment, R A is unsubstituted propyl. In an embodiment, R A is unsubstituted n-propyl. In an embodiment, R A It is an unsubstituted isopropyl group. In the implementation scheme, R A It is an unsubstituted butyl. In the implementation scheme, RA It is an unsubstituted n-butyl. In the implementation scheme, R A It is an unsubstituted isobutyl group. In the implementation scheme, R A It is unsubstituted tert-butyl.

[0271] In an embodiment, substituted R 10 (e.g., substituted alkyl and / or substituted heteroalkyl) is substituted by at least one substituent, a size-restricted substituent, or a lower substituent; wherein if the substituted R 10 The substituent is replaced by a plurality of groups selected from substituents, size-restricted substituents, and lower substituents; each substituent, size-restricted substituent, and / or lower substituent may optionally be different. In an embodiment, when R 10 When substituted, it is replaced by at least one substituent. In the implementation, when R 10 When substituted, it is substituted with at least one substituent of limited size. In an embodiment, when R 1 When O is substituted, it is substituted with at least one lower substituent.

[0272] In an embodiment, R 10 It is hydrogen, CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2CI, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4 or C1-C2), or substituted or unsubstituted heteroalkyl (e.g., 2 to 8-membered, 2 to 6-membered, 4 to 6-membered, 2 to 3-membered or 4 to 5-membered).

[0273] In an embodiment, R 10 It is hydrogen. In the implementation scheme, R 10 It is an unsubstituted C1-C4 alkyl group. In the embodiments, R 10 is unsubstituted methyl. In an embodiment, R 10 is an unsubstituted ethyl group. In an embodiment, R 10 is unsubstituted propyl. In an embodiment, R 10 is unsubstituted n-propyl. In an embodiment, R 10 It is an unsubstituted isopropyl group. In the implementation scheme, R 10 It is an unsubstituted butyl. In the implementation scheme, R 10 It is an unsubstituted n-butyl. In the implementation scheme, R 10 It is an unsubstituted isobutyl group. In the implementation scheme, R 10 It is unsubstituted tert-butyl.

[0274] In an embodiment, when R1 and R 6 The substituted ring formed when the substituents are linked (e.g., substituted heterocycloalkyl and / or substituted heteroaryl) is substituted with at least one substituent, size-restricted substituent, or lower substituent; wherein when R 1 and R 6 The substituted ring formed when the substituents are linked is substituted with a plurality of groups selected from substituents, size-limited substituents, and lower substituents; each substituent, size-limited substituent, and / or lower substituent may optionally be different. In an embodiment, when R 1 and R 6 When the substituted ring formed when the substituents are attached is substituted, it is substituted with at least one substituent. 1 and R 6 When the substituted ring formed when the substituents are attached is substituted, it is substituted with at least one substituent of limited size. 1 and R 6 When a substituted ring formed when substituents are linked is substituted, it is substituted with at least one lower substituent.

[0275] In an embodiment, R 1 and R 6 Substituents connect to form substituted or unsubstituted 3- to 8-membered heterocyclic alkyl groups. In the embodiments, R 1 and R 6 The substituents are linked to form a substituted or unsubstituted pyrrolidinyl group. 1 and R 6 The substituents are linked to form an unsubstituted pyrrolidinyl group. In one embodiment, R 1 and R 6 The substituents are linked to form a substituted or unsubstituted 5- to 6-membered heteroaryl group.

[0276] In an embodiment, when R 8 and R 10 When substituents are linked, the resulting substituted ring (e.g., a substituted heterocyclic alkyl group and / or a substituted heteroaryl group) is substituted by at least one substituent, a size-restricted substituent, or a lower substituent; wherein when R 8 and R 10 The substituted ring formed when the substituents are linked is substituted with a plurality of groups selected from substituents, size-limited substituents, and lower substituents; each substituent, size-limited substituent, and / or lower substituent may optionally be different. In an embodiment, when R 8 and R 10 When the substituted ring formed when the substituents are attached is substituted, it is substituted with at least one substituent. 8 and R 10When a substituent ring formed during substituent linkage is substituted, it is substituted by at least one size-constrained substituent. In an embodiment, when R 8 and R 10 When a substituted ring formed when substituents are linked is substituted, it is substituted with at least one lower substituent.

[0277] In an embodiment, R 8 and R 10 Substituents connect to form substituted or unsubstituted 3- to 8-membered heterocyclic alkyl groups. In the embodiments, R 8 and R 10 The substituents are linked to form a substituted or unsubstituted pyrrolidinyl group. 8 and R 10 The substituents are linked to form an unsubstituted pyrrolidinyl group. In one embodiment, R 8 and R 10 The substituents are linked to form a substituted or unsubstituted 5- to 6-membered heteroaryl group.

[0278] In an embodiment, the compound has the formula:

[0279]

[0280] In an embodiment, the compound has the formula:

[0281]

[0282] In an embodiment, the compound has the formula:

[0283]

[0284] In an embodiment, the compound has the formula:

[0285]

[0286] In an embodiment, the compound has the formula:

[0287]

[0288] In an embodiment, the compound has the formula:

[0289]

[0290] In an embodiment, the compound has the formula:

[0291]

[0292] In an embodiment, the compound has the formula:

[0293]

[0294] In an embodiment, the compound has the formula:

[0295]

[0296] In an embodiment, the compound has the formula:

[0297]

[0298] In an embodiment, the compound has the formula:

[0299]

[0300] In an embodiment, the compound has the formula:

[0301]

[0302] In an embodiment, the compound has the formula:

[0303]

[0304] In an embodiment, the compound has the formula:

[0305]

[0306] In an embodiment, the compound has the formula:

[0307]

[0308] In an embodiment, the compound has the formula:

[0309]

[0310] In an embodiment, the compound has the formula:

[0311]

[0312] In an embodiment, the compound has the formula:

[0313]

[0314] In an embodiment, the compound has the formula:

[0315]

[0316] In an embodiment, the compound has the formula:

[0317]

[0318] In the implementation scheme, the compound has the following formula:

[0319]

[0320] In the implementation scheme, the compound has the following formula:

[0321]

[0322] In the implementation scheme, the compound has the following formula:

[0323]

[0324] In the implementation scheme, the compound has the following formula:

[0325]

[0326] In the implementation scheme, the compound has the following formula:

[0327]

[0328] In the implementation scheme, the compound has the following formula:

[0329]

[0330] In the implementation scheme, the compound has the following formula:

[0331]

[0332] In the implementation scheme, the compound has the following formula:

[0333]

[0334] In the implementation scheme, the compound has the following formula:

[0335]

[0336] In the implementation scheme, the compound has the following formula:

[0337]

[0338] In the implementation scheme, the compound has the following formula:

[0339]

[0340] In the implementation scheme, the compound has the following formula:

[0341]

[0342] In the embodiments, the compound is a compound as described herein, included in the embodiments. In the embodiments, the compound is a compound described herein (e.g., in the Examples section, figures, tables, or claims).

[0343] In one aspect, an oligonucleotide having the formula:

[0344] B.L 1 、R 1 、R 2 、R 4 、R 6 、R 7 、R 8 、R 9 and R 10 As described herein, it is included in the implementation scheme.

[0345] L 5 It is a bivalent oligonucleotide linker containing 4 to 40 nucleotides.

[0346] L 50 It is a bond, -NH-, -O-, -S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene (e.g., C1-C8, C1-C6, C1-C4, or C1-C2), substituted or unsubstituted heteroalkylene (e.g., 2 to 8-membered, 2 to 6-membered, 4 to 6-membered, 2 to 3-membered, or 4 to 5-membered), substituted or unsubstituted cycloalkylene (e.g., C3-C8, C3-C6, C4-C6, or C5-C6), substituted or unsubstituted heteroalkylene (e.g., 3 to 8-membered, 3 to 6-membered, 4 to 6-membered, 4 to 5-membered, or 5 to 6-membered), substituted or unsubstituted arylene (e.g., C6-C 10 or phenylene), or a substituted or unsubstituted heteroarylene (e.g., 5- to 10-membered, 5- to 9-membered, or 5- to 6-membered).

[0347] R 50 is the detectable portion.

[0348] R 30 Yes-OR 30A or -OP(NR 3B R 3C )-OR 3A .

[0349] R 30A It is hydrogen, a 3' closed part, or a monovalent oligonucleotide part.

[0350] R 3A 、R 3Band R 3C and -CHCl, -CBr, -CF, -CI, -CHCl, -CHBr, -CHF, -CHI, -CHCl, -CHBr, -CHF, -CHI, -CN, -OH, -NH, -COOH, -CONH, -OCCl, -OCF, -OCBr, -OCI, -OCHCl, -OCHBr, -OCHI, -OCHF, -OCHCl, -OCHBr, -OCHI, -OCHF, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0351] In an embodiment, the oligonucleotide has the formula:

[0352] B.L 1 、L 5 、L 50 、R 2 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0353] In an embodiment, the oligonucleotide has the formula:

[0354] B.L 1 、L 5 、L 50 、R 2 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0355] In an embodiment, the oligonucleotide has the formula:

[0356] B.L 1 、L 5 、L 50 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0357] In an embodiment, the oligonucleotide has the formula:

[0358] B.L 1 、L 5 、L 50 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0359] In an embodiment, the oligonucleotide has the formula:

[0360] L 1 , L 5 , L 50 、R 2 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0361] In an embodiment, the oligonucleotide has the formula:

[0362] L 1 , L 5 , L 50 、R 2 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0363] In an embodiment, the oligonucleotide has the formula:

[0364] L 1 , L 5 , L 50 、R 2 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0365] In an embodiment, the oligonucleotide has the formula:

[0366] L 1 , L 5 , L 50 、R 2 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0367] In an embodiment, the oligonucleotide has the formula:

[0368] L 1 , L 5 , L 50 、R 2 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0369] In an embodiment, the oligonucleotide has the formula:

[0370] L 1 、L 5 、L 50 、R 2 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0371] In an embodiment, the oligonucleotide has the formula:

[0372] L 1 、L 5 、L 50 、R 2 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0373] In an embodiment, the oligonucleotide has the formula:

[0374] L 1 、L 5 、L 50 、R 2 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0375] In an embodiment, the oligonucleotide has the formula:

[0376] L 1 、L 5 、L 50 、R 2 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0377] In an embodiment, the oligonucleotide has the formula:

[0378] L 1 、L 5 、L 50 、R 2 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0379] In an embodiment, the oligonucleotide has the formula:

[0380] L 1 、L 5 、L 50 、R 2、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0381] In an embodiment, the oligonucleotide has the formula:

[0382] L 1 、L 5 、L 50 、R 2 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0383] In an embodiment, the oligonucleotide has the formula:

[0384] L 1 、L 5 、L 50 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0385] In an embodiment, the oligonucleotide has the formula:

[0386] L 1 、L 5 、L 50 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0387] In an embodiment, the oligonucleotide has the formula:

[0388] L 1 、L 5 、L 50 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0389] In an embodiment, the oligonucleotide has the formula:

[0390] L 1 、L 5 、L 50 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0391] In an embodiment, the oligonucleotide has the formula:

[0392] L1 、L 5 、L 50 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0393] In an embodiment, the oligonucleotide has the formula:

[0394] L 1 、L 5 、L 50 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0395] In an embodiment, the oligonucleotide has the formula:

[0396] L 1 、L 5 、L 50 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0397] In an embodiment, the oligonucleotide has the formula:

[0398] L 1 、L 5 、L 50 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0399] In an embodiment, the oligonucleotide has the formula:

[0400] L 1 、L 5 、L 50 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0401] In an embodiment, the oligonucleotide has the formula:

[0402] L 1 、L 5 、L 50 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0403] In an embodiment, the oligonucleotide has the formula:

[0404] L 1 , L 5 , L 50 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0405] In an embodiment, the oligonucleotide has the formula:

[0406] L 1 , L 5 , L 50 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0407] In an embodiment, L 5 It contains 11 to 30 nucleotides. In the implementation scheme, L... 5 It contains 19 to 23 nucleotides. In the implementation scheme, L... 5 It contains 4 to 14 nucleotides. In the implementation scheme, L... 5 It contains 6 to 12 nucleotides.

[0408] In this implementation, the nucleotide is a DNA nucleotide. In this implementation, the nucleotide is an RNA nucleotide.

[0409] In an embodiment, substituted L 50 (e.g., substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene and / or substituted heteroarylene) is substituted with at least one substituent, size-restricted substituent, or lower substituent; wherein if the substituted L 50 is substituted with a plurality of groups selected from substituents, size-limited substituents and lower substituents; each substituent, size-limited substituent and / or lower substituent may optionally be different. In an embodiment, when L 50 When substituted, it is replaced by at least one substituent. In the embodiment, when L 50 When substituted, it is replaced by at least one size-restricted substituent. In the embodiment, when L 50 When substituted, it is substituted with at least one lower substituent.

[0410] In an embodiment, L 50 It is a substituted 2 to 10-membered heteroalkylene group. In the embodiments, L 50 It is an oxo-substituted 2- to 10-membered heteroalkylene group. In the embodiments, L 50 yes

[0411] In an embodiment, R 50 It is the fluorescent portion (e.g., as described herein). In the implementation, R 50 is a monovalent form of FAM, a monovalent form of VIC, a monovalent form of ABY, a monovalent form of JUN, a monovalent form of AF647, a monovalent form of Cy5, a monovalent form of AF676, or a monovalent form of Cy5.5. In an embodiment, R 50 This is the unit price for Alexa Fluor 488. In the implementation plan, R... 50 is the monovalent form of FAM. In an embodiment, R 50 is a monovalent form of TET. In an embodiment, R 50 is the monovalent form of HEX. In the embodiment, R 50 is the monovalent form of JOE. In an embodiment, R 50 is the monovalent form of VIC. In an embodiment, R 50 is a monovalent form of Cy3. In an embodiment, R 50 is the monovalent form of Alexa Fluor 555. In one embodiment, R 50 is the monovalent form of NED. In an embodiment, R 50 is a monovalent form of Alexa Fluor 546. In one embodiment, R 50 is a monovalent form of TAMRA. In an embodiment, R 50 This is the unit price format for Dye 3.0. In the implementation plan, R... 50 is the monovalent form of ABY. In an embodiment, R 50 is a monovalent form of PET. In an embodiment, R 50 It is in the form of a unit price of Dy33.5. In the implementation plan, R... 50 This is the unit price for Alexa Fluor 568. In the implementation plan, R... 50 is a monovalent form of ROX. In an embodiment, R5 0 is the monovalent form of TX Red. In an embodiment, R 50 This is the unit price for Alexa Fluor594. In the implementation plan, R... 50 This is the unit price format for Dye 4.0. In the implementation plan, R... 50 is the monovalent form of JUN. In an embodiment, R 50 is a monovalent form of d-ROX. In an embodiment, R 50 This is the unit price for Alexa Fluor 647. In the implementation plan, R... 50 is a monovalent form of Cy5. In an embodiment, R 50It is the unit price of AF676. In the implementation plan, R 50 It is the unit price format of Cy5.5.

[0412] In an embodiment, R 30 Yes - OR 30A In an embodiment, R 30 is -OH. In an embodiment, R 30 Yes -OP(NR) 3B R 3C )-OR 3A , where R 3A 、R 3B and R 3C As described herein, included in the embodiments. In the embodiments, R 30 yes

[0413] In an embodiment, R 30A It is hydrogen. In the implementation scheme, R 30A It is a 3' closed portion. In the implementation plan, R 30A It is the monovalent oligonucleotide moiety.

[0414] In embodiments, the 3′ blocking moiety is a monovalent form of dideoxycytidine (3′ddC). In embodiments, the 3′ blocking moiety is a monovalent form of dideoxyadenosine (ddA). In embodiments, the 3′ blocking moiety is a 3′ reversed dT. In embodiments, the 3′ blocking moiety is a 3′ amino modifier. In embodiments, the 3′ blocking moiety is a monovalent form of QSY7. In embodiments, the 3′ blocking moiety is a monovalent form of QSY21. In embodiments, the 3′ blocking moiety is a monovalent form of QSY9. In embodiments, the 3′ blocking moiety is a monovalent form of BHQ1. In embodiments, the 3′ blocking moiety is a monovalent form of BHQ2. In embodiments, the 3′ blocking moiety is a monovalent form of BHQ3. In embodiments, the 3′ blocking moiety is a monovalent form of Dabcyl. In embodiments, the 3′ blocking moiety is a monovalent form of Dabsy1. In embodiments, the 3′ blocking moiety is a monovalent form of Eclipse. In embodiments, the 3′ blocking moiety is a monovalent form of BBQ-650. In an embodiment, the 3' blocking moiety is a monovalent form of Iowa Black RQ. In an embodiment, the 3' blocking moiety is a monovalent form of Iowa Black FQ. In an embodiment, the 3' blocking moiety is In the implementation plan, the 3' closed portion is In the implementation plan, the 3' closed portion is In the implementation plan, the 3' closed portion is

[0415] In an embodiment, the oligonucleotide has the formula:

[0416]

[0417] III. How to Use

[0418] In one aspect, a method for forming a double-stranded nucleic acid is provided, the method comprising contacting a target oligonucleotide with a nucleic acid probe containing a quencher moiety (e.g., an internal quencher moiety) to form a double-stranded nucleic acid; wherein the nucleic acid probe has the following formula:

[0419] (XIX) or its salts. B, L 1 、L 5 、L 50 、R 2 、R 4 and R 50 As described herein, it is included in the implementation scheme. 30 Yes-OR 30A , where R 30A is a monovalent oligonucleotide moiety. Q is a quencher moiety (e.g., as described herein). In embodiments, Q is an internal quencher moiety (e.g., as described herein).

[0420] In embodiments, the quencher moiety (e.g., an internal quencher moiety) is a monovalent form of QSY7. In embodiments, the quencher moiety (e.g., an internal quencher moiety) is In embodiments, the quencher moiety (e.g., an internal quencher moiety) is In embodiments, the quencher moiety (e.g., an internal quencher moiety) is a monovalent form of QSY21. In embodiments, the quencher moiety (e.g., an internal quencher moiety) is In embodiments, the quencher moiety (e.g., an internal quencher moiety) is In embodiments, the quencher moiety (e.g., an internal quencher moiety) is a monovalent form of QSY9. In embodiments, the quencher moiety (e.g., an internal quencher moiety) is In embodiments, the quencher moiety (e.g., an internal quencher moiety) is In embodiments, the quencher moiety (e.g., an internal quencher moiety) is a monovalent form of BHQ1. In embodiments, the quencher moiety (e.g., an internal quencher moiety) is In embodiments, the quencher moiety (e.g., an internal quencher moiety) is In embodiments, the quencher moiety (e.g., an internal quencher moiety) is a monovalent form of BHQ2. In embodiments, the quencher moiety (e.g., an internal quencher moiety) is In embodiments, the quencher moiety (e.g., an internal quencher moiety) is In embodiments, the quencher moiety (e.g., an internal quencher moiety) is a monovalent form of BHQ3. In embodiments, the quencher moiety (e.g., an internal quencher moiety) is In embodiments, the quencher moiety (e.g., an internal quencher moiety) is In an embodiment, the quencher moiety (e.g., an internal quencher moiety) is a monovalent form of Dabcyl. In an embodiment, the quencher moiety (e.g., an internal quencher moiety) is In embodiments, the quencher moiety (e.g., an internal quencher moiety) is In an embodiment, the quencher moiety (e.g., an internal quencher moiety) is a monovalent form of Dabsyl. In an embodiment, the quencher moiety (e.g., an internal quencher moiety) is In embodiments, the quencher moiety (e.g., internal quencher moiety) is a monovalent form of Eclipse. In embodiments, the quencher moiety (e.g., internal quencher moiety) is a monovalent form of In embodiments, the quencher moiety (e.g., an internal quencher moiety) is In embodiments, the quencher moiety (e.g., internal quencher moiety) is a monovalent form of BBQ-650. In embodiments, the quencher moiety (e.g., internal quencher moiety) is In one embodiment, the quencher portion (e.g., the internal quencher portion) is in unit price form of Iowa Black RQ. In another embodiment, the quencher portion (e.g., the internal quencher portion) is in unit price form of Iowa Black FQ. In yet another embodiment, the quencher portion (e.g., the internal quencher portion) described herein can be used in any of the methods described herein.

[0421] In embodiments, Q is a quencher moiety (eg, an internal quencher moiety) having the formula:

[0422] R 1 、R 6 、R 7 、R 8 、R 9 and R 10 As described herein, it is included in the implementation scheme.

[0423] In one embodiment, the method further includes contacting the double-stranded nucleic acid with a polymerase having exonuclease activity to cleave the quencher portion (e.g., an internal quencher portion) from the detectable portion. In another embodiment, the method further includes detecting a signal generated by the detectable portion. In another embodiment, the detectable portion is a fluorescent portion (e.g., as described herein). In another embodiment, the method further includes amplifying the target oligonucleotide using PCR. In another embodiment, the PCR is quantitative PCR. In another embodiment, the PCR is digital PCR.

[0424] In one aspect, a method for detecting nucleic acids in a sample is provided, the method comprising: (i) providing a reaction mixture comprising: at least a portion of the sample; a first probe detectably labeled with a first label, configured to generate a first fluorescent signal; and a second probe detectably labeled with a second label, configured to generate a second fluorescent signal, wherein the first and second probes have different sequences, wherein the first and second labels are identical and / or generate substantially the same fluorescence; and wherein the second probe has the following formula:

[0425] (XIX) or its salts, wherein Q, B, L 1 、L 5 、L 50 、R 2 and R 4 As described herein, it is included in the implementation scheme; R 30 Yes-OR 30A , where R 30A is the monovalent oligonucleotide portion; R 50 (ii) subjecting the reaction mixture to an amplification process comprising a first set of reaction conditions and a second set of reaction conditions, the first set of reaction conditions being different from the second set of reaction conditions; and (iii) determining the presence of a first and / or second nucleic acid target in the sample by measuring fluorescence signals during the first set of reaction conditions, the fluorescence signals during the first set of reaction conditions being correlated with a specific interaction or lack thereof between the first probe and the first nucleic acid target, measuring fluorescence signals during the second set of reaction conditions, the fluorescence signals during the second set of reaction conditions being correlated with a specific interaction or lack thereof between the first probe and the first nucleic acid target, and estimating the presence and / or amount of the first and second nucleic acid targets.

[0426] In one aspect, a method for detecting nucleic acids in a sample is provided, the method comprising: (i) providing a reaction mixture comprising: at least a portion of the sample; a first probe detectably labeled with a first label, configured to generate a first fluorescent signal indicating the presence or absence of a first nucleic acid target; and a second probe detectably labeled with a second label, configured to generate a second fluorescent signal indicating the presence or absence of a second nucleic acid target, wherein the first and second probes have different sequences, wherein the first and second labels are identical and / or generate substantially the same fluorescence; and wherein the second probe has the following formula:

[0427] (XIX) or its salts, wherein Q, B, L 1 , L 5 , L 50 、R 2 and R 4 As described herein, it is included in the implementation scheme; R 30 Yes - OR 30A , where R 30A It is the monovalent oligonucleotide moiety; R 50 (ii) subjecting the reaction mixture to an amplification process comprising a first set of reaction conditions and a second set of reaction conditions, the first set of reaction conditions being different from the second set of reaction conditions; and (iii) determining the presence and / or amount of the first and / or second nucleic acid targets in the reaction mixture by measuring a first total fluorescence signal comprising any first fluorescence signal (if present) during the first set of reaction conditions, measuring a second total fluorescence signal comprising any first fluorescence signal (if present) and any second fluorescence signal (if present) during the second set of reaction conditions, and estimating the first fluorescence signal and / or the second fluorescence signal based on the first total fluorescence signal and the second total fluorescence signal.

[0428] In the implementation scheme, both the first probe and the second probe are excited at the same wavelength, and / or both the first probe and the second probe are excited during the detection of their respective first fluorescence signal and second fluorescence signal.

[0429] In the implementation scheme, measuring the fluorescence signal during the first set of reaction conditions includes measuring a combined signal including a first fluorescence signal and a second fluorescence signal during the first set of reaction conditions to obtain a first total fluorescence signal; measuring the fluorescence signal during the second set of reaction conditions includes measuring a combined signal including a first fluorescence signal and a second fluorescence signal during the second set of reaction conditions to obtain a second total fluorescence signal; and estimating the presence and / or amount of the first nucleic acid target and the second nucleic acid target includes estimating the first fluorescence signal and / or the second fluorescence signal based on the first total fluorescence signal and the second total fluorescence signal.

[0430] In embodiments, the degree to which the second fluorescent signal differs between the first set of reaction conditions and the second set of reaction conditions is greater than the degree to which the first fluorescent signal differs between the first set of reaction conditions and the second set of reaction conditions.

[0431] In an embodiment, the first total fluorescence value includes (i) fluorescence from the first label, which is free and unquenched in the reaction mixture and emitted due to the first label being cleaved after hybridization of the first probe with the first amplicon, and (ii) background fluorescence from the second label, and the second total fluorescence value is based on (i) fluorescence from the first label, which is free and unquenched in the reaction mixture and emitted due to the first label being cleaved after hybridization of the first probe with the first amplicon, and (ii) fluorescence from the second label, which is emitted above the background fluorescence of the second label due to hybridization of the second probe with the second amplicon.

[0432] In embodiments, the method further comprises calculating the amount of the first nucleic acid target based on the first fluorescent signal; and calculating the amount of the second nucleic acid target based on the second fluorescent signal.

[0433] In embodiments, when the first nucleic acid target is present in the reaction mixture, the first fluorescent signal is above background levels during both the first set of reaction conditions and the second set of reaction conditions. In embodiments, when the second nucleic acid target is present in the reaction mixture, the second fluorescent signal is above background levels during the second set of reaction conditions but not during the first set of reaction conditions.

[0434] In one embodiment, the first set of reaction conditions includes a first measurement temperature for measuring a first fluorescence signal, and the second set of reaction conditions includes different second measurement temperatures for measuring a second fluorescence signal. In one embodiment, the first measurement temperature and the second measurement temperature differ by at least about 10°C or more. In one embodiment, the first measurement temperature and the second measurement temperature differ by at least about 15°C or more. In one embodiment, the first measurement temperature and the second measurement temperature differ by at least about 20°C or more. In one embodiment, the first measurement temperature and the second measurement temperature differ by at least about 25°C or more. In one embodiment, the first measurement temperature and the second measurement temperature differ by at least about 30°C or more. In one embodiment, at least one of the first or second measurement temperature is a denaturation temperature at which the DNA in the reaction mixture is denatured. In one embodiment, the denaturation temperature is about 90°C or higher.

[0435] In embodiments, the reaction mixture is subjected to a plurality of amplification cycles, each cycle comprising a first set of reaction conditions and a second set of reaction conditions. In embodiments, the amplification process comprises thermal cycling.

[0436] In one embodiment, the first set of reaction conditions includes a denaturation step at a first temperature of thermal cycling. In another embodiment, the second set of reaction conditions includes an annealing and / or extension step (“annealing / extension step”) at a second temperature of thermal cycling, which is lower than the first temperature.

[0437] In embodiments, the first probe is a cleavable probe. In embodiments, when the cleavable probe is cleaved during the annealing / extension step, the first fluorescent signal increases. In embodiments, the first probe comprises a fluorophore (e.g., as described herein) and a quencher (e.g., as described herein), and wherein the first probe is configured such that fluorescence from the fluorophore is quenched by the quencher until the probe is cleaved during the annealing / extension step of the amplification process. In embodiments, the first probe is a TaqMan probe.

[0438] In the implementation scheme, the second probe is a non-cleavable probe. In the implementation scheme, when the second probe is single-chain, L... 5 In one embodiment, Q and R 50 They are spaced apart so that when the second probe is single-stranded, R 50 It was quenched, and when the second probe was incorporated into the double-stranded amplicon, R 50 In an embodiment, Q and R 50 They are all located at or near the stem ring portion of the second probe.

[0439] In embodiments, the reaction mixture further comprises: a first primer pair complementary to a first nucleic acid target of a nucleic acid or its complement, the first nucleic acid target being configured to produce a first amplicon with which the first probe can hybridize; and a second primer pair complementary to a second nucleic acid target of a nucleic acid or its complement, the second nucleic acid target being configured to produce a second amplicon with which the second probe can hybridize. In embodiments, the second primer pair comprises a primer having a tail. In embodiments, the tail forms the 5' end of the primer having the tail. In embodiments, the second probe can hybridize to the tail or its complement.

[0440] In embodiments, the amplification process utilizes a series of thermal cycling steps including at least three different target temperatures. In embodiments, the amplification process includes a denaturation temperature and a plurality of different annealing / extension temperatures that vary throughout the amplification process. In embodiments, a first series of denaturation steps and annealing / extension steps are performed at a first annealing / extension temperature, and wherein a second series of denaturation steps and annealing / extension steps are performed at a second, different annealing / extension temperature. In embodiments, the first annealing / extension temperature is higher than the second annealing / extension temperature. In embodiments, the first series of denaturation steps and annealing / extension steps are cycled more times than the second series of denaturation steps and annealing / extension steps.

[0441] In one embodiment, the amplification process further includes a third series of denaturation and annealing / extension steps using a third annealing / extension temperature. In another embodiment, the third annealing / extension temperature is the same as the first annealing / extension temperature. In yet another embodiment, the third series of denaturation and annealing / extension steps are cycled more times than the first series of denaturation and annealing / extension steps. In yet another embodiment, the denaturation temperature is the same for each series of denaturation and annealing / extension steps.

[0442] In one embodiment, the second primer pair comprises a tailed primer and a tailless primer, wherein the tailed primer and the tailless primer are provided at different concentrations. In another embodiment, the tailless primer is provided at a higher concentration than the tailed primer. In another embodiment, the tailless primer is provided at a concentration of approximately 2X (2 times) to approximately 30X (30 times) of the tailed primer concentration. In another embodiment, the tailless primer is provided at a concentration of approximately 5X to approximately 25X of the tailed primer concentration. In another embodiment, the tailless primer is provided at a concentration of approximately 10X to approximately 20X of the tailed primer concentration. In another embodiment, the second probe is provided at a concentration different from the concentrations of the tailed primer and the tailless primer. In another embodiment, the second probe is provided at a concentration greater than the tailed primer concentration. In another embodiment, the second probe is provided at a concentration lower than the tailless primer concentration. In another embodiment, the second probe is provided at a concentration of approximately 2X to approximately 10X of the tailed primer concentration. In another embodiment, the second probe is provided at a concentration of approximately 3X to approximately 7.5X of the tailed primer concentration.

[0443] In an embodiment, the melting temperature (T m ) and the T of the second probe m are within about 8°C of each other. In embodiments, the melting temperature (T m ) and the T of the second probe m are within about 6°C of each other. In embodiments, the melting temperature (T m ) and the T of the second probe m are within about 4°C of each other. In embodiments, the melting temperature (T m ) and the T of the second probe m The temperature difference between them is within about 2°C.

[0444] In one embodiment, the amplification process is cycled multiple times between two target temperatures. In another embodiment, the amplification process is cycled at least 5% of its cycles between two target temperatures. In yet another embodiment, the amplification process is cycled at least 10% of its cycles between two target temperatures. In yet another embodiment, the amplification process is cycled at least 15% of its cycles between two target temperatures. In yet another embodiment, the amplification process is cycled at least 20% of its cycles between two target temperatures. In yet another embodiment, the amplification process is cycled at least 25% of its cycles between two target temperatures. In yet another embodiment, the amplification process is cycled at least 30% of its cycles between two target temperatures. In yet another embodiment, the amplification process is cycled at least 35% of its cycles between two target temperatures. In yet another embodiment, the amplification process is cycled at least 40% of its cycles between two target temperatures. In yet another embodiment, the amplification process is cycled at least 45% of its cycles between two target temperatures. In yet another embodiment, the amplification process is cycled at least 50% of its cycles between two target temperatures. In one embodiment, the amplification process is cycled at least 55% of its cycles between two target temperatures. In another embodiment, the amplification process is cycled at least 60% of its cycles between two target temperatures. In yet another embodiment, the amplification process is cycled at least 65% of its cycles between two target temperatures. In yet another embodiment, the amplification process is cycled at least 70% of its cycles between two target temperatures. In yet another embodiment, the amplification process is cycled at least 75% of its cycles between two target temperatures. In yet another embodiment, the amplification process is cycled at least 75% of its cycles between two target temperatures. In yet another embodiment, the amplification process is cycled at least 80% of its cycles between two target temperatures. In yet another embodiment, the amplification process is cycled at least 85% of its cycles between two target temperatures. In yet another embodiment, the amplification process is cycled at least 90% of its cycles between two target temperatures. In yet another embodiment, the amplification process is cycled at least 95% of its cycles between two target temperatures.

[0445] In the embodiments, the reaction mixture is added to or formed within multiple reaction volumes, and the amplification process is a digital PCR (dPCR) process.

[0446] In embodiments, measuring the fluorescent signal during the first set of reaction conditions comprises a first endpoint measurement under the first set of reaction conditions, and wherein measuring the fluorescent signal during the second set of reaction conditions comprises a second endpoint measurement under the second set of reaction conditions.

[0447] In the implementation scheme, estimating the presence and / or quantity of the first nucleic acid target and the second nucleic acid target includes: classifying multiple reaction volumes based on signals measured at a first endpoint and signals measured at a second endpoint; and based on the classification, determining a count of reaction volumes in which the first probe exhibits activity and a count of reaction volumes in which the second probe exhibits activity.

[0448] In one aspect, a method for detecting nucleic acid in a sample is provided, the method comprising: providing a reaction mixture comprising a primer pair complementary to a nucleic acid target or its complement for producing an amplicon and a non-cleavable probe configured to hybridize to the amplicon, the probe comprising a detectable label configured to provide a fluorescent signal corresponding to the amount of amplicon produced, subjecting the reaction mixture to an amplification process to produce the amplicon, wherein the label produces fluorescence and the probe is not cleaved during the amplification process, and wherein the amplification process utilizes a series of thermal cycling steps comprising at least three different target temperatures; and measuring the fluorescent signal from the probe; wherein the probe has the formula:

[0449] (XIX) or its salts, wherein Q, B, L 1 , L 5 , L 50 、R 2 and R 4 As described herein, it is included in the implementation scheme; R 30 Yes - OR 30A , where R 30A It is the monovalent oligonucleotide moiety; R 50 It is a detectable marker.

[0450] In the implementation scheme, the method also includes quantifying the amount of nucleic acid target based on measured fluorescence signals.

[0451] In one embodiment, the probe includes a stem-loop portion, which, when the probe is single-chained, can form a stem-loop structure. In another embodiment, Q and R... 50 They are spaced apart, so that when the probe is single-stranded, R 50 It was quenched, but when the probe was incorporated into the double-stranded amplicon R... 50 It is enabled. In the implementation, Q and R 50 They are all placed at or near the stem ring portion of the probe.

[0452] In one embodiment, the primer pair includes a primer having a tail. In another embodiment, the tail forms the 5' end of the primer having the tail. In yet another embodiment, the probe is configured to hybridize with the tail or its complement. In yet another embodiment, the 3' portion of the probe is configured to hybridize with the tail or its complement.

[0453] In embodiments, the amplification process includes a denaturation temperature and a plurality of different annealing / extension temperatures that vary throughout the amplification process. In embodiments, a first series of denaturation steps and annealing / extension steps are performed at a first annealing / extension temperature, and wherein a second series of denaturation steps and annealing / extension steps are performed at a second, different annealing / extension temperature. In embodiments, the first annealing / extension temperature is higher than the second annealing / extension temperature. In embodiments, the first series of denaturation steps and annealing / extension steps are cycled more times than the second series of denaturation steps and annealing / extension steps.

[0454] In one embodiment, the amplification process further includes a third series of denaturation and annealing / extension steps using a third annealing / extension temperature. In another embodiment, the third annealing / extension temperature is the same as the first annealing / extension temperature. In yet another embodiment, the third series of denaturation and annealing / extension steps are cycled more times than the first series of denaturation and annealing / extension steps. In yet another embodiment, the denaturation temperature is the same for each series of denaturation and annealing / extension steps.

[0455] In an embodiment, the primer pair comprises a tailed primer and a tailless primer, and wherein the tailed primer and the tailless primer are provided at different concentrations. In an embodiment, the tailless primer is provided at a higher concentration than the tailed primer. In an embodiment, the tailless primer is provided at a concentration of about 2X to about 30X the concentration of the tailed primer. In an embodiment, the tailless primer is provided at a concentration of about 5X to about 25X the concentration of the tailed primer. In an embodiment, the tailless primer is provided at a concentration of about 10X to about 20X the concentration of the tailed primer. In an embodiment, the probe is provided at a concentration different from the concentration of the tailed primer and the concentration of the tailless primer. In an embodiment, the probe is provided at a concentration greater than the concentration of the tailed primer. In an embodiment, the probe is provided at a concentration lower than the concentration of the tailless primer. In an embodiment, the probe is provided at a concentration of about 2X to about 10X the concentration of the tailless primer. In an embodiment, the probe is provided at a concentration of about 3X to about 7.5X the concentration of the tailless primer.

[0456] In one aspect, a method for detecting nucleic acid in a sample is provided, the method comprising: providing a reaction mixture comprising a primer pair targeting a nucleic acid target for producing an amplicon, the primer pair comprising a tailed primer and a tailless primer provided at different concentrations, and a non-cleavable probe configured to hybridize with the amplicon, the probe comprising a detectable label configured to produce a fluorescent signal corresponding to the amount of the amplicon produced, subjecting the reaction mixture to an amplification process to produce the amplicon, wherein the probe produces fluorescence without being cleaved during the amplification process; and measuring the fluorescent signal from the probe; wherein the probe has the formula:

[0457] (XIX) or its salts, wherein Q, B, L1 、L 5 、L 50 、R 2 and R 4 As described herein, it is included in the implementation scheme; R 30 Yes-OR 30A , where R 30A is the monovalent oligonucleotide portion; R 50 is a detectable label.

[0458] In some embodiments, the tailless primer is provided at a higher concentration than the tailed primer. In some embodiments, the tailless primer is provided at a concentration of about 2X to about 30X of the tailed primer concentration. In some embodiments, the tailless primer is provided at a concentration of about 5X to about 25X of the tailed primer concentration. In some embodiments, the tailless primer is provided at a concentration of about 10X to about 20X of the tailed primer concentration. In some embodiments, the probe is provided at a concentration different from both the tailed and tailless primer concentrations. In some embodiments, the probe is provided at a concentration greater than the tailed primer concentration. In some embodiments, the probe is provided at a concentration lower than the tailless primer concentration. In some embodiments, the probe is provided at a concentration of about 2X to about 10X of the tailless primer concentration. In some embodiments, the probe is provided at a concentration of about 3X to about 7.5X of the tailless primer concentration.

[0459] In one aspect, a method for detecting the presence or amount of a first and / or second target in a reaction mixture is provided, the method comprising: comprising a first probe and a second probe, wherein the first probe can specifically interact with the first target and comprises a first label that can produce a first detectable signal, and the second probe can specifically interact with the second target and comprises a second label that can produce a second detectable signal; wherein the second probe has the following formula:

[0460] (XIX) or its salts, wherein Q, B, L 1 、L 5 、L 50 、R 2 and R4 are as described herein, including in embodiments; R 30 Yes-OR 30A , where R 30A is a monovalent oligonucleotide moiety; and R 50is a second label; allowing the first probe and the second probe to specifically interact with any first target and second target in the reaction mixture, respectively; measuring a first total signal through a filter under a first set of conditions, wherein the first total signal includes the first detectable signal and the second detectable signal from the first label and the second label, and wherein under the first set of conditions, the first detectable signal is increased due to the specific interaction of the first probe with the first target, but the second detectable signal is not increased due to the specific interaction of the second probe with the second target; measuring a second total signal through the same filter under a second set of conditions, wherein the second total signal includes the first detectable signal and the second detectable signal from the first label and the second label, and wherein under the second set of conditions, the second detectable signal is increased due to the specific interaction of the second probe with the second target; and assessing the presence or amount of the first target and / or the second target by estimating the first detectable signal and the second detectable signal based on both the first total signal and the second total signal.

[0461] In one embodiment, the first label and the second label are identical and / or produce substantially the same fluorescence. In another embodiment, the difference in the second fluorescence signal between the first set of conditions and the second set of conditions is greater than the difference in the first fluorescence signal between the first set of conditions and the second set of conditions.

[0462] In embodiments, the first probe is a cleavable probe. In embodiments, when the cleavable probe is cleaved, the first detectable signal increases. In embodiments, the first probe comprises a fluorophore (e.g., as described herein) and a quencher (e.g., as described herein), and wherein the first probe is configured such that the fluorescence from the fluorophore is quenched by the quencher until the probe is cleaved. In embodiments, the first probe is a TaqMan probe.

[0463] In the implementation scheme, the second probe is a non-cleavable probe. In the implementation scheme, when the second probe is single-chain, L... 5 A stem-ring structure is formed. In the implementation scheme, R 50 is a fluorophore, and Q and R 50 They are spaced apart so that when the second probe is single-stranded, R 50 It was quenched, and when the second probe was incorporated into the double-stranded amplicon, R 50 In an embodiment, Q and R 50 They are all located at or near the stem ring portion of the second probe.

[0464] In an embodiment, the melting temperature (T m ) and the T of the second probe m are within about 8°C of each other. In embodiments, the melting temperature (Tm ) and the T of the second probe m are within about 6°C of each other. In embodiments, the melting temperature (T m ) and the T of the second probe m are within about 4°C of each other. In embodiments, the melting temperature (T m ) and the T of the second probe m The temperature difference between them is within about 2°C.

[0465] In embodiments, the first set of conditions includes a first measurement temperature for measuring the first fluorescent signal, and the second set of conditions includes a different second measurement temperature for measuring the second fluorescent signal. In embodiments, the first measurement temperature and the second measurement temperature differ by at least about 10°C or more. In embodiments, the first measurement temperature and the second measurement temperature differ by at least about 15°C or more. In embodiments, the first measurement temperature and the second measurement temperature differ by at least about 20°C or more. In embodiments, the first measurement temperature and the second measurement temperature differ by at least about 25°C or more. In embodiments, the first measurement temperature and the second measurement temperature differ by at least about 30°C or more. In embodiments, at least one of the first measurement temperature or the second measurement temperature is a denaturation temperature at which the DNA in the reaction mixture denatures. In embodiments, the denaturation temperature is about 90°C or above.

[0466] In embodiments, the method further comprises thermally cycling the reaction mixture between the two target temperatures for a plurality of cycles. In embodiments, the thermal cycling cycles between the two target temperatures for at least 5% of the cycles. In embodiments, the thermal cycling cycles between the two target temperatures for at least 10% of the cycles. In embodiments, the thermal cycling cycles between the two target temperatures for at least 15% of the cycles. In embodiments, the thermal cycling cycles between the two target temperatures for at least 20% of the cycles. In embodiments, the thermal cycling cycles between the two target temperatures for at least 25% of the cycles. In embodiments, the thermal cycling cycles between the two target temperatures for at least 30% of the cycles. In embodiments, the thermal cycling cycles between the two target temperatures for at least 35% of the cycles. In embodiments, the thermal cycling cycles between the two target temperatures for at least 40% of the cycles. In embodiments, the thermal cycling cycles between the two target temperatures for at least 45% of the cycles. In embodiments, the thermal cycling cycles between the two target temperatures for at least 50% of the cycles. In embodiments, the thermal cycling cycles between the two target temperatures for at least 55% of the cycles. In embodiments, the thermal cycling cycles between the two target temperatures for at least 60% of the cycles. In embodiments, the thermal cycle cycles between two target temperatures for at least 65% of the cycles. In embodiments, the thermal cycle cycles between two target temperatures for at least 70% of the cycles. In embodiments, the thermal cycle cycles between two target temperatures for at least 75% of the cycles. In embodiments, the thermal cycle cycles between two target temperatures for at least 80% of the cycles. In embodiments, the thermal cycle cycles between two target temperatures for at least 85% of the cycles. In embodiments, the thermal cycle cycles between two target temperatures for at least 90% of the cycles. In embodiments, the thermal cycle cycles between two target temperatures for at least 95% of the cycles.

[0467] IV. Intra-channel multiplexing (multiplexing using probes labeled with spectral similarity)

[0468] Figure 1A The emission spectra of various fluorescent dyes commonly used for nucleic acid detection assays are shown. As described herein, conventional multiple determinations assign each dye to a separate target, and then determine the presence and / or amount of each target by measuring the fluorescent signal, for example, in a separate detection channel, each of which corresponds to the emission wavelength of the corresponding dye. As shown, there is usually a large amount of overlap in the emission spectrum of the dye. Although the purpose of selecting multiple dyes is to minimize spectral overlap, the limited nature of emission spectra actually limits the number of separate dyes that can be combined in the same multiple determination, and therefore limits the number of different targets that can be detected and / or measured.

[0469] The embodiments described herein address one or more of the aforementioned problems by providing multiple detectable signals within the same detection channel, each detectable signal associated with a different assay target or group of targets. The multiple detectable signals can be individually resolved and analyzed independently, thereby allowing the detection and / or quantification of each target. By allowing the assay of multiple targets within the same detection channel, the disclosed embodiments advantageously increase the diversity of multiplex assays without relying on additional dyes and the associated problem of increased spectral overlap. Similarly, the embodiments described herein advantageously reduce the number of individual dyes required in multiplex assays without compromising assay diversity.

[0470] Figure 2A This is a schematic overview of a method for detecting multiple target nucleic acids within the same detection channel by providing different types of first and second probes, varying the reaction mixture conditions, and measuring the total signal obtained under each set of conditions. As shown, the first probe is designed to specifically interact ("bind") with a first target. The first probe contains a first label capable of generating a first labeled signal. The second probe is designed to specifically interact with a second target different from the first target. The second probe contains a second label capable of generating a second labeled signal.

[0471] In some embodiments, the first and second markers are identical. For example, the first and second markers may contain the same fluorescent dye. In some embodiments, the first and second markers may be different, but are still designed to produce substantially the same signal (e.g., substantially the same fluorescence). For example, the first and second markers may contain chemically different dyes that function to emit fluorescent signals with similar wavelengths. In some embodiments, the first and second marker signals are measured using the same filter arrangement in the detection instrument. The first and second marker signals are therefore measured within the same detection channel.

[0472] The first probe and the second probe can be provided in identical reaction mixture, and it is allowed to interact specifically with any first target and the second target in the reaction mixture respectively.As shown in the figure, reaction mixture is subjected to at least two groups of different reaction conditions.The first probe is designed so that during the first group of conditions and the second group of conditions, the first label produces the first label signal, and its degree is relevant (for example, proportional) to the amount of the specific interaction between the first probe and the first target.On the contrary, the second probe is designed so that the second label produces the second label signal during the second group of conditions rather than during the first group of conditions, and its degree is relevant (for example, proportional) to the amount of the specific interaction between the second probe and the second target. In other words, under the first group of conditions, the first label signal increases due to the specific interaction of the first probe and the first target, but the second label signal does not increase due to the specific interaction of the second probe and the second target, and under the second group of conditions, the second label signal increases due to the specific interaction of the second probe and the second target.

[0473] During the first set of conditions, the second label will not produce "substantial fluorescence", and therefore the second label signal will not be substantially different from the background (i.e., baseline) level of fluorescence in the reaction mixture. That is, although there may be a certain non-zero level of signal produced by the second label during the first set of conditions, the second label signal will generally remain below the threshold that separates background fluorescence from meaningful signal. As described above, this threshold can vary depending on the specific test protocol and application requirements.

[0474] In at least some embodiments, when both the first target and the second target are present in the reaction mixture, the second marker signal will differ more between the first set of conditions and the second set of conditions than the first marker signal will differ between the first set of conditions and the second set of conditions. Thus, while the first marker signal may differ somewhat between the first set of conditions and the second set of conditions, that difference will generally be less than the difference in the second marker signal between the first set of conditions and the second set of conditions.

[0475] The differences in how the first marker signal and the second marker signal respond to different sets of conditions can be exploited to enable separation of the first marker signal and the second marker signal even when the first marker signal and the second marker signal are within the same detection channel. For a given detection channel (e.g., for a given filter arrangement), the total signal during a first set of conditions ("first total signal") is measured, and the total signal during a second set of conditions ("second total signal") is measured.

[0476] During the first set of conditions, the total signal will be essentially equal to the first marker signal. That is, the first total signal mainly consists of the first marker signal, while the contribution from the second marker signal is negligible. During the second set of conditions, the total signal will include a combination of the first and second marker signals. Therefore, the first and second marker signals can be analyzed independently based on the first and second total signals. For example, the first marker signal can be determined based on the first total signal, and the second marker signal can be analyzed by subtracting the first total signal from the second total signal.

[0477] In some implementations, the first marker signal is directly equal to the first total signal. In other implementations, the first marker signal is determined as a function of the first total signal. In some implementations, this function is linear. For example, as mentioned above, even when the amount of the first target remains unchanged, the first marker signal may differ slightly between the first set of conditions and the second set of conditions. In some applications, the first marker signal under the second set of conditions may better correspond to a standard curve that equates the first marker signal to the amount of the first target. Therefore, estimating the first marker signal as a function of the first total signal, rather than as directly equal to the first total signal, allows the calculated first marker signal to be closer to the signal to be measured under the second set of conditions (i.e., a second marker signal without any interference).

[0478] In some embodiments, the function for converting the first total signal into the first labeled signal is determined by comparing the first labeled signal under a first set of conditions with the first labeled signal under a second set of conditions without any interaction between the second probe and the second target. The first labeled signals under the first and second sets of conditions are typically correlated with each other according to a linear function. Therefore, a simple multiplier can be used to convert the first total signal into the first labeled signal. Once such a linear function is determined, it can be used in subsequent measurements without the need for additional comparisons of the first labeled signal under the first set of conditions and under the second set of conditions without a second probe having a second target. In some embodiments, the function for converting the first total signal into the first labeled signal may be nonlinear. In some embodiments, the function / correlation is determined when the expected number of cleavage probes is the same. That is, the first total signal of cycle "n" is compared with the second total signal of cycle "n-1".

[0479] As described in more detail below, the first and second probes have different mechanisms of action, achieving different signal responses to the first and second sets of conditions depending on the probe type. Advantageously, unlike some prior methods, the ability to resolve individual signals associated with each different probe type does not need to depend on the different melting temperatures of the probes. Therefore, although the first and second probes may have different melting temperatures, they do not necessarily need to have substantially different melting temperatures to allow for efficient resolution of individual signals. For example, in some embodiments, the melting temperature of the first probe (T...) m ) (conventionally defined as the temperature at which 50% of the strands are in double-stranded form and 50% are single-stranded) and the T of the second probe. m The temperature difference between them is within approximately 8°C, 6°C, 4°C, or 2°C. In some embodiments, under either the first or second set of conditions, both probes bind (e.g., hybridize) to their respective targets. Optionally, under either the first or second set of conditions, neither probe substantially binds (e.g., hybridizes) to its respective target. In one example, under the first set of conditions, both the first and second probes substantially bind (e.g., hybridize) to their respective targets, while under the second set of conditions, neither the first nor the second probe substantially binds (e.g., hybridizes) to their respective targets. In another example, under the first set of conditions, both the first and second probes substantially bind (e.g., hybridize) to their respective targets, while under the second set of conditions, both the first and second probes substantially bind (e.g., hybridize) to their respective targets.

[0480] Figure 2B is a graph showing that when the reaction mixture is cycled between a first set of reaction conditions and a second set of reaction conditions and when both the first target and the second target are present in the reaction mixture, Figure 2A Graph over time of the signal response of the method outlined in . Cycling of conditions can include, for example, thermal cycling in a nucleic acid amplification reaction, where a first set of reaction conditions represents a denaturation step at a first temperature of the thermal cycle, and a second set of reaction conditions includes an annealing and / or extension step ("annealing / extension step") at a second temperature (lower than the first temperature) of the thermal cycle.

[0481] As shown, under the second set of reaction conditions, both the first marker signal and the second marker signal increased. Under the first set of reaction conditions, the first marker signal remained approximately the same as at the end of the previous cycle (although, as described above, it may have changed slightly), while the second marker signal decreased to a level similar to the baseline signal level.

[0482] As shown in the figure, the first marker signal and the second marker signal accumulate and increase at each successive occurrence of the second set of conditions. This is the result of the additional specific interactions in the reaction mixture between the first probe and the first target and the additional specific interactions in the reaction mixture between the second probe and the second target. However, when the first marker signal moves from the end of one cycle to the beginning of another cycle (that is, when moving from the second set of conditions at the end of the cycle to the first set of conditions at the beginning of the subsequent cycle), the second marker signal returns to a level close to the baseline at the beginning of each cycle (that is, at each occurrence of the first set of conditions).

[0483] While the embodiments described herein primarily focus on intra-channel multiplexing, the disclosed intra-channel multiplexing can be combined with inter-channel multiplexing to further increase the diversity of an assay. For example, an assay can be designed using a variety of different dyes (and therefore a variety of different detection channels), wherein two or more different channels each include a variety of detectable signals that can be resolved using the methods described herein.

[0484] V. Fractable and Non-Fractable Probes

[0485] In an embodiment, the first probe is a "cleavable" probe. The first probe can be designed such that as a result of hybridization of the first probe to the first target, the first label is separated from the first probe (and, for example, released from the corresponding quencher). Thus, once released, the first label continues to contribute to the total signal in the reaction mixture. The first probe can be a TaqMan probe, for example, which undergoes cleavage due to the 5' to 3' exonuclease activity of a DNA polymerase during extension of the target molecule to which the probe is hybridized. TaqMan probes are described in U.S. Patent Nos. 4,889,818, 5,079,352, 5,210,015, 5,436,134, 5,487,972, 5,658,751, 5,210,015, 5,487,972, 5,538,848, 5,618,711, 5,677,152, 5,723,591, 5,773,258, 5,789,224, 5,8 and 7,445,900, all of which are hereby incorporated by reference herein.

[0486] In this implementation, the second probe is a "non-cleavable" probe. The second labeling of the non-cleavable probe is intended to maintain correlation with the probe throughout the assay and to alter the level of the generated signal based on the probe configuration rather than the release of the label. The second probe could be an EF probe, for example, one that quenches the label when in a single-chain configuration but allows the signal when incorporated into a double-chain molecule.

[0487] In embodiments, the second probe has the formula:

[0488] Or its salt, wherein Q is the quencher portion (e.g., the internal quencher portion) having the following formula:

[0489] B is a divalent nucleobase; L 1 is a divalent linker; L 5 is a divalent oligonucleotide linker comprising 4 to 40 nucleotides; L 50 It is a bond, -NH-, -O-, -S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O-), substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 50 Is the second mark; R 30 Yes-OR 30A ; R 30A is the monovalent oligonucleotide portion; R 2 Is it hydrogen or -OR 2A ; R 4 is hydrogen or unsubstituted methyl, or R 2 and R 4 Substituents connect to form substituted or unsubstituted heterocyclic alkyl groups; R 1 and R 10 Independently, it is hydrogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, substituted or unsubstituted alkyl groups, or substituted or unsubstituted heteroalkyl groups; R 6 、R 7 、R 8 and R 9are independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3R A -SO2NH2, -NHNH2, -ONH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCBr3, -OCF3, -OCI3, -OCH2Cl, -OCH2Br, -OCH2F, -OCH2I, -OCHCl2, -OCHBr2, -OCHF2, -OCHI2, -SF5, -N3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 1 and R 6 can be connected to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group; and R 8 and R 10 It can be linked to form substituted or unsubstituted heterocyclic alkyl groups or substituted or unsubstituted heteroaryl groups; and R 2A and R A Independently, it is hydrogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In the embodiments, Q, B, L 1 , L 5 , L 50 、R 1 、R 2 、R 4 、R 6 、R 7 、R 8 、R 9 、R 10 、R 30 and R 50 As described herein, it is included in the implementation scheme.

[0490] Figure 3A The activities of cleavable TaqMan probe 102 and non-cleavable EF probe 112 during the annealing, extension, and denaturation steps of thermal cycling are shown. As shown, TaqMan probe 102 hybridizes with its corresponding target 104 during the annealing phase. During extension, at primer 103 positioned upstream of probe 102, the 5′ to 3′ exonuclease activity of DNA polymerase cleaves the TaqMan probe tag 106 from the remainder of probe 102, thereby separating it from the corresponding TaqMan probe quencher 108. This results in a corresponding increase in fluorescence signal. During denaturation, tag 106 remains free in the reaction mixture solution and thus continues to contribute to the total fluorescence signal.

[0491] The EF probe 112 comprises an EF probe label 116 and an EF probe quencher 118, which remain close to each other when the probe 112 is in a single-stranded configuration. Therefore, when the EF probe is in a single-stranded configuration, the fluorescence signal from the label 112 is essentially quenched. During the annealing and extension phases, the EF probe 112 hybridizes with its corresponding target template 114 and extends to form an extended probe amplicon 113. The extension of the target template 114 then forms a complement 115 to the extended probe amplicon 113. The resulting double-stranded amplicon 119 forces the label 116 away from the quencher 118 to a distance sufficient to allow fluorescence emission. During denaturation, the extended probe amplicon 113 separates from its complement 115. Upon returning to the single-stranded configuration, the label 116 and the quencher 118 are brought back into proximity, and fluorescence is quenched again.

[0492] Figure 3B This is a graph showing the fluorescence signals from the TaqMan and EF probes over time during the thermal cycling of the amplification process. The temperature of the thermal cycling can be varied depending on the specific application requirements. For example, the denaturation step can be performed at a temperature of about 90°C or higher, such as about 95°C, and the annealing / extension step can be performed at a lower temperature, such as about 50°C to about 75°C. Here, the first set of reaction conditions corresponds to the denaturation step, and the second set of reaction conditions corresponds to the annealing / extension step.

[0493] While the currently preferred embodiments cycle between a denaturation step and a combined annealing / extension step (i.e., the amplification process cycles between two target temperatures), other embodiments may include separate annealing and extension steps. In such embodiments, the temperature may vary between the annealing and extension steps. For example, the extension step may be performed at a temperature higher than the annealing temperature. In embodiments, the amplification process is cycled between the two target temperatures for at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the amplification process.

[0494] Figure 3B As shown, the fluorescence signal associated with the TaqMan probe increases during the extension phase and then remains at a similar level in the denaturation phase of the next cycle, while the fluorescence signal associated with the EF probe increases during the extension phase but decreases to baseline once the subsequent denaturation phase reaches the target denaturation temperature.

[0495] In an embodiment, the first set of reaction conditions (e.g., denaturation conditions) includes a first measurement temperature for measuring the first marking signal, and the second set of reaction conditions (e.g., annealing / extending conditions) includes different second measurement temperatures for measuring the first and second marking signals. In an embodiment, the first and second measurement temperatures differ by at least about 10°C or more, about 15°C or more, about 20°C or more, about 25°C or more, or about 30°C or more. The first measurement temperature may be a target denaturation temperature of about 90°C or higher, such as about 95°C, and the second measurement temperature may be a target annealing / extending temperature, such as about 50°C to about 75°C.

[0496] VI. EF probe template formation

[0497] Figure 4A The process of forming a target template 114 that the EF probe 112 can hybridize with using a tailed primer 122 that is specific to the nucleic acid target 124 is illustrated. The tailed primer 122 includes a tail 126 and a target-specific portion 128. Figure 4B Examples of more detailed views of the tailed primer 122 as the forward primer, the target-specific primer 123 paired with the tailed primer 122 as the reverse primer, and the EF probe 112 are shown.

[0498] As shown, in the first stage, target-specific portion 128 hybridizes to target 124. Extension of target-specific portion 128 forms tailed amplicon 125. Primer 123, which pairs with tailed primer 122, extends the complement 114 of tailed amplicon 125. It is this complement that forms target template 114. As shown, target template 114 includes tail complement portion 127.

[0499] In the second stage, the EF probe 112 hybridizes to the target template 114, and amplification can continue, as Figure 3A As shown. Figure 4A As shown, the EF probe 112 includes a probe tail 117 that has substantial homology to the tail 126 and is therefore complementary to the tail complement portion 127 of the target template 114. Extension of the probe 112 and the target template 114 forms a double-stranded amplicon 119. In the third stage, a primer 123, shown here as being paired with the tailed primer 122, can also be used as the primer 123 paired with the EF probe 112 to achieve the formation of the double-stranded amplicon 119, as shown. Figure 3A shown.

[0500] like Figure 4B As shown, tail 126 may form the 5′ end of tailed primer 122. EF probe 112 may include a stem-loop portion, wherein stem portion 110 is on either side of loop portion 111, configured to form a stem-loop structure when EF probe 112 is single-stranded. For example, marker 116 may be located on one side of the stem-loop portion, and quencher 118 may be located on the opposite side of the stem-loop portion, such that when the stem-loop structure is formed, marker 116 and quencher 118 are close together, but when EF probe 112 is constrained to a more linear configuration (e.g., when a double-stranded amplicon is incorporated), the marker and quencher are spaced further apart.

[0501] In the illustrated embodiment, marker 116 is located at or near the 5′ end of EF probe 112, and quencher 118 is located at the 3′ end of marker 116. In other embodiments, the positions of marker 116 and quencher 118 may be reversed. Preferably, as shown, the stem-loop portion is located at the 5′ end of probe tail 117, such that the stem-loop portion remains at the end of the amplicon generated by the extension of EF probe 112, thereby making stem-loop structure formation (when single-stranded) less likely to be damaged.

[0502] In addition to or as an alternative to the EF probes described herein, some embodiments may include other labeled oligonucleotides that produce increased fluorescence when incorporated into double-stranded amplicons (relative to when in a single-stranded state). For example, LUX TM The primer contains an internal fluorophore that is quenched by a hairpin structure located 5' of the fluorophore. Like the EF probe, when incorporated into the double-stranded amplicon, the LUX TMThe primers provide increased fluorescence, and the hairpin structure is linearized. Furthermore, any primers or probes described herein may include one or more locked nucleic acids (LNAs) known in the art.

[0503] In some embodiments, tailed primer 122 and corresponding (tailless) primer 123 are provided at different concentrations. For example, primer 123 can be provided at a higher concentration than tailed primer 122. For example, primer 123 can be provided at a concentration of about 2X to about 30X the concentration of tailed primer 122, or about 5X to about 25X the concentration of tailed primer 122, or about 10X to about 20X the concentration of tailed primer 122. Because primer 123 can play a role in (1) driving the formation of target template 114 (e.g., Figure 4A ) and (2) driving the formation of a complement 115 of the extended probe amplicon 113 (as shown in FIG. Figure 3A ), so providing it at a higher concentration than the corresponding tailed primer 122 can beneficially balance the reaction and help drive overall reaction efficiency.

[0504] In some embodiments, EF probe 112 is provided at a concentration that is different from the concentration of tailed primer 122 and / or the concentration of primer 123. For example, EF probe 112 can be provided at a concentration that is greater than the concentration of tailed primer 122 and less than the concentration of primer 123. In some embodiments, EF probe 112 is provided at a concentration that is from about 2X to about 20X the concentration of tailed primer 122, or from about 3X to about 15X the concentration of tailed primer 122. As described above, providing a relatively high concentration of primer 123 helps drive the overall efficiency of the reaction. Compared to tailed primer 122, providing EF probe 112 at a higher concentration than tailed primer 122, but not necessarily higher than primer 123, drives more of the associated amplification toward EF probe 112, while still allowing primer 123 to be the primary driver of reaction efficiency.

[0505] In addition to or as an alternative to using a "universal" EF probe with a probe tail 117, other embodiments include and / or utilize EF probes having a target-specific portion instead of a probe tail 117. Such EF probes can hybridize directly to a target template and therefore do not need to follow Figure 4A The two-stage process shown is used to generate a target template 114 having a tail complement portion 127. In such an embodiment, the probe tail 117 of the EF probe 112 is replaced with a target-specific portion that hybridizes directly to the target 124. The process is otherwise similar to Figure 3A That is, after extension of the EF probe, a subsequent round of annealing / extension will extend the complementary strand to form a double-stranded amplicon that separates the fluorophore and quencher to allow for the generation of a fluorescent signal.

[0506] Figure 4CShown is a three-stage thermal cycling method that can be used during an amplification process involving an EF probe and, optionally, a TaqMan probe. Figure 4C The amplification process illustrated can be used in conjunction with any other methods disclosed herein. The illustrated amplification process includes a first stage with a first target annealing / extension temperature, a second stage with a different second annealing / extension temperature, and a third stage with a third annealing / extension temperature. In this embodiment, the third annealing / extension temperature is the same as the first annealing / extension temperature. Other embodiments may include a third annealing / extension temperature different from both the first and second annealing / extension temperatures.

[0507] Therefore, the amplification process shown includes denaturation temperatures that vary throughout the amplification process and multiple different annealing / extension temperatures. This type of amplification process advantageously provides an initial stage (stage 1) for the initial formation of the target template 114, an intermediate stage (stage 2) for the increased interaction between the EF probe 112 and the target template 114 to form the initially extended probe amplicon 113, and a later stage (stage 3) in which further amplification involving probe amplicon 113 and 115 can proceed.

[0508] As shown in the figure, the first annealing / extending temperature can be higher than the second annealing / extending temperature. The first series of modification steps and annealing / extending steps (in stage 1) are repeated more times than the second series of modification steps and annealing / extending steps (in stage 2). The third series of modification steps and annealing / extending steps (in stage 3) can be repeated more times than the first series of modification steps and annealing / extending steps. The modification temperature of each series of modification steps and annealing / extending steps can be the same.

[0509] Therefore, stages 1 and 2 serve as preloading stages, primarily generating target template 114 (in stage 1), followed by a lower annealing / extension temperature (in stage 2) for at least one cycle to allow increased interaction between EF probe 112 and target template 114. Subsequently, multiple amplification cycles can be performed at a third annealing / extension temperature to drive amplification primarily involving EF probe 112, primer 123, and / or their extensions, probe amplicones 113 and 115. Thus, most amplification cycles typically occur during stage 3.

[0510] VII. Digital PCR

[0511] PCR is an in vitro enzymatic method for the synthesis of specific DNA sequences using two oligonucleotide primers that hybridize to opposite strands and flank a region of interest in the target DNA. A series of repeated reaction steps, involving template denaturation, primer annealing, and extension of the annealed primers by a DNA polymerase, results in the exponential accumulation of specific fragments, whose termini are defined by the primers. PCR can selectively enrich specific DNA sequences over several orders of magnitude.

[0512] Digital polymerase chain reaction (dPCR) is a specific type of PCR that can be used to directly quantify target nucleic acids. In dPCR, the reaction mixture is partitioned into many small reaction volumes (i.e., partitions) so that the target nucleic acid is in some, but not all, of the reaction volumes / partitions. The reaction volumes are subjected to thermal cycling, and the proportion of "positive" partitions that produce a signal (usually a fluorescent signal) indicating the presence of the target is determined. Quantification is based on the application of Poisson statistics, using the number of negative / non-reactive reaction volumes and assuming a Poisson distribution to determine the number of initial copies distributed across all reaction volumes.

[0513] The features and principles of other amplification processes described herein are also generally applicable to dPCR processes. Therefore, although the description of this section provides a more detailed disclosure of dPCR embodiments, the disclosure provided elsewhere herein is also applicable to dPCR embodiments. Embodiments including dPCR can utilize various partitioning mechanisms or devices known in the art or that may be developed in the future. For example, some conventional dPCR systems utilize multiple droplets encapsulated by an oil phase to form multiple partitions / reaction volumes. Other embodiments can utilize microchamber arrays. An example of such a system is the QuantStudio Absolute Q system available from Thermo Fisher Scientific, which uses a microfluidic array plate to partition / partition samples.

[0514] In some dPCR embodiments, the reaction mixture is fully formed before being partitioned into multiple reaction volumes. In alternative embodiments, one or more components of the reaction mixture can be preloaded onto or into the reaction volumes. For example, probes and / or primers can be coated onto the walls of a microchamber, and then samples and / or other components of the reaction mixture can be added to the microchamber to form multiple reaction mixtures in each reaction volume.

[0515] Figure 5A is a schematic overview of a method for detecting multiple target nucleic acids within the same detection channel using a dPCR process. Although embodiments including real-time / quantitative PCR (qPCR) typically monitor amplification during the reaction, dPCR typically includes an endpoint measurement to count and determine the number of "positive" partitions. Therefore, Figure 5AIt is shown that under a first set of conditions (e.g., denaturing conditions, such as about 95° C.) and a second set of conditions (e.g., annealing / extension conditions, such as about 65° C.), partitions in which the first probe and the first target are present will be shown as positive (+) at the end of the reaction. On the other hand, partitions in which the second probe and the second target are present will be shown as negative (-) under the first set of conditions, but will be shown as positive (+) under the second set of conditions.

[0516] This is similar to Figure 2A The scheme shown, and Figure 5A It is shown that the same principle can be applied to endpoint measurements as in dPCR.As described in other embodiments, the first probe can be a cleavable probe, such as a TaqMan probe, and the second probe can be a non-cleavable probe, such as an EF probe.

[0517] Figure 5B How the signal (for example, at the endpoint measurement) of dPCR subregion is shown according to whether the first probe, the second probe or both are active in the subregion during the reaction and change.As shown in the figure, if only the first probe provides signal, then under the first group of conditions and the second group of conditions, the subregion will be positive (+).If only the second probe provides signal, then the subregion will be negative (-) under the first group of conditions and will be positive (+) under the second group of conditions.If the first probe and the second probe both provide signal, then the subregion will be positive (+) under the first group of conditions, and will be highly positive (++) under the second group of conditions.Certainly, if both probe types do not provide signal, then under any one group of conditions, the subregion will all be negative (-).

[0518] The total count of partitions that are positive for the first probe (and therefore estimated to be positive for the first target) is determined by counting the number of partitions that are positive (+) or highly positive (++) under both sets of conditions. The total count of partitions that are positive for the second probe (and therefore estimated to be positive for the second target) is determined by counting (i) the number of partitions that are positive (+) under the second set of conditions but negative (-) under the first set of conditions and adding this to (ii) the number of partitions that are highly positive (++) under the second set of conditions. These partition counts can be calculated or estimated by plotting the endpoint signal under the first set of conditions against the endpoint signal under the second set of conditions and identifying clusters. For example, see Figure 8 The concentrations of the first and second targets in the sample can then be estimated using standard dPCR techniques.

[0519] Thus, a method for determining the presence and / or amount of multiple targets using the intra-channel multiplexing feature described herein in a dPCR application includes the following steps: preparing a reaction mixture comprising a first probe type (e.g., a TaqMan probe) and a second probe type (e.g., an EF probe) that target corresponding first and second nucleic acid targets; loading / partitioning the sample into a plurality of reaction volumes; measuring the endpoint signal of the reaction volumes under a first set of reaction conditions (e.g., denaturation conditions, such as approximately 95°C); measuring the endpoint signal of the reaction volumes under a second set of reaction conditions (e.g., annealing / extension conditions, such as approximately 65°C); classifying the reaction volumes according to the signal characteristics measured at the endpoint measurement; determining or estimating the counts of each probe type based on the classified reaction volumes (i.e., the counts of the reaction volumes in which the first probe type is active and the counts of the reaction volumes in which the second probe type is active); and determining or estimating the presence and / or amount of the first and second nucleic acid targets based on the counts.

[0520] VIII. Additional Markings / Dye Details

[0521] Exemplary detectable markers that can be used in the embodiments described herein include, for example:

[0522] Fluoresceins (e.g., 5-carboxy-2,7-dichlorofluorescein, 5-carboxyfluorescein (5-FAM), 6-JOE, 6-carboxyfluorescein (6-FAM), VIC, FITC, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE)), 5 and 6-carboxy-1,4-dichloro-2',7'-dichlorofluorescein (TET), 5 and 6-carboxy-1,4-dichloro-2',4',5',7'-tetrachlorofluorescein, HEX, PET, NED, Oregon Green (e.g., 488, 500, 514));

[0523] Pyrenes; (e.g., Cascade Blue; Alexa Fluor 405);

[0524] Coumarins (e.g., Pacific Blue, Atto 425, Alexa Fluor 350, Alexa Fluor 430);

[0525] Anthocyanin dyes; (e.g., Cy dyes, such as Cy3, Cy3.18, Cy3.5, Cy5, Cy5.18, Cy5.5, Cy7);

[0526] Rhodamines; (e.g., 110, 123, B, B 200, BB, BG, B Extra, 5 and 6-carboxytetramethylrhodamine (5-TAMRA, 6-TAMRA), 5 and 6-carboxyrhodamine 6G, Lissamine, Lissamine Rhodamine B, Rhod-2, ROX (6-carboxy-X-rhodamine), 5 and 6-ROX (carboxy-X-rhodamine), Sulforhodamine B and C, Sulforhodamine G Extra, 5 and 6TAMRA (6-carboxytetramethyl-rhodamine), (TRITC), ABY, JUN, LIZ, RAD, RXJ, Texas Red; and Texas Red-X);

[0527] ALEXA FLUOR fluorophores (a broad class that includes many dye types, such as cyanines) (e.g., Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 676, 680, 700, 750);

[0528] FRET donor / acceptor pairs (e.g., fluorescein / fluorescein, fluorescein / rhodamine, fluorescein / cyanine, rhodamine / cyanine, fluorescein / Alexa Fluor, Alexa Fluor / rhodamine); and other types of dyes known to those skilled in the art.

[0529] Fluorophore labels can be associated with quenchers such as dark fluorescence quenchers (DFQ), black hole quenchers (BHQ), IowaBlack, QSY7, QSY21 quenchers, Dabsyl and Dabcel sulfonate / carboxylate quenchers, and MGB-NFQ quenchers. For example, fluorophore labels can also include sulfonate derivatives of fluorescein dyes with SO3 replacing the carboxylate group, phosphoramidite forms of fluorescein, and / or phosphoramidite forms of Cy5.

[0530] IX. Additional Amplification Details

[0531] Amplification products produced using one or more embodiments described herein can be produced, detected, and / or analyzed on any suitable platform. In embodiments, the nucleic acid target can be single-stranded, double-stranded, or any other nucleic acid molecule of any size or conformation. Amplification processes as described herein can include PCR (see, e.g., U.S. Patent No. 4,683,202). In embodiments, PCR is qPCR. In embodiments, PCR is endpoint PCR. In some embodiments, PCR is dPCR.

[0532] In the embodiments, the amplification process includes RT-PCR. Disclosed methods may include, for example, subjecting the target nucleic acid to a reverse transcription reaction prior to PCR amplification. In some embodiments, the amplification process includes a one-step RT-PCR (e.g., in a single container or reaction volume) in which one or more reverse transcriptases are used in combination with one or more DNA polymerases.

[0533] Optionally, certain qPCR assays can be plated in individual wells of a single array or multi-well plate, such as, for example, a TaqMan Array Card (see, e.g., Thermo Fisher Scientific, Waltham, MA; catalog numbers 4346800 and 4342265) or a MicroAmp multi-well (e.g., 96-well, 384-well) reaction plate (see, e.g., Thermo Fisher Scientific, Waltham, MA; catalog numbers 4346906, 4366932, 4306737, 4326659, and N8010560). Optionally, the different qPCR assays present in different wells of an array or plate can be dried or freeze-dried in situ, and the array or plate can be stored or shipped before use. In some embodiments, the concepts described herein can be used in in situ hybridization applications that are not necessarily associated with PCR.

[0534] Other amplification methods, such as loop-mediated isothermal amplification (“LAMP”) and other isothermal methods, may also be used in the assay implementation described herein.

[0535] The components described herein for enabling in-channel reuse may be provided in the kit along with one or more additional components to enable the amplification process. Such components may include, for example, dNTPs, DNA polymerase, amplification buffer / reagent, master mixture components known in the art, and other components known in the art for enabling or assisting nucleic acid amplification.

[0536] X. Specific Implementation of the Computer System

[0537] In an embodiment, at least a portion of the methods described herein may be implemented using one or more computer systems. In an embodiment, the techniques discussed herein are represented by computer-executable instructions that may be stored on one or more hardware storage devices. The computer-executable instructions may be executable by one or more processors to implement (or configure a system to implement) the disclosed techniques. In some embodiments, the system may be configured to send the computer-executable instructions to a remote device to configure the remote device to perform the disclosed techniques.

[0538] In one example implementation, the computer system includes one or more processors and a memory storing one or more instructions that, when executed by the one or more processors, cause the one or more processors to perform the following processes: at multiple time points during one or more cycles of an amplification process, obtaining fluorescence signal data associated with a composite fluorescence signal from at least a first probe type containing a first fluorophore and a second probe type containing a second fluorophore, the second fluorophore having substantially overlapping spectral features with the first fluorophore and / or producing the same or substantially identical signal, the first probe type and the second probe type being different in thermal and / or temporal characteristics; and during the one or more cycles of the amplification process, determining fluorescence signal data associated with a fluorescence signal from a given probe type of at least the first probe type and the second probe type, based at least in part on the fluorescence signal data associated with the composite fluorescence signal and the thermal and / or temporal characteristics of one or more of the at least first probe type and the second probe type.

[0539] In an implementation, using fluorescence signal data associated with a composite fluorescence signal and first fluorescence signal data as input to generate fluorescence signal data associated with a fluorescence signal from a given probe type includes: generating transformed first fluorescence signal data by applying a transformation (e.g., linear) to the first fluorescence signal data; and modifying the fluorescence signal data associated with the composite fluorescence signal with the transformed first fluorescence signal data to generate fluorescence signal data associated with a fluorescence signal from a given probe type.

[0540] In an implementation scheme, when executed by one or more processors, one or more instructions further cause one or more processors to perform the following processes: quantizing a first target associated with a first probe type based at least on first fluorescence signal data; and quantizing a second target associated with a second probe type based at least on generated fluorescence signal data associated with a fluorescence signal from a given probe type.

[0541] Some implementations include one or more computer-readable media storing one or more instructions that, when executed by one or more processors of at least one computing device, cause the one or more processors to perform the aforementioned process as described herein or other computer-implemented processes.

[0542] Systems for implementing the disclosed embodiments may include various components such as, by way of non-limiting example, processors, memories, sensors, I / O systems, communication systems, and the like. A processor may include one or more sets of electronic circuits comprising any number of logic units, registers, and / or control units for executing computer-readable instructions (e.g., instructions forming a computer program). Such computer-readable instructions may be stored in a storage device. The storage device may include physical system memory and may be volatile, non-volatile, or some combination thereof. Furthermore, the storage device may include a local storage device, a remote storage device (e.g., accessible via a communication system or otherwise), or some combination thereof.

[0543] Furthermore, the system may include or communicate with an I / O system. The I / O system may include any type of input or output device, such as, but not limited to, a touch screen, a mouse, a keyboard, a controller, a speaker, and / or the like. For example, the I / O system may include a display system, which may include any number of display panels, optical devices, laser scanning display components, and / or other components.

[0544] The disclosed embodiments may also include physical and other computer-readable media for carrying or storing computer-executable instructions and / or data structures. Such computer-readable media can be any available media that can be accessed by a general-purpose or special-purpose computer system. Computer storage media (also referred to as "hardware storage devices") are computer-readable hardware storage devices based on RAM, flash memory, phase-change memory ("PCM") or other types of memory, such as RAM, ROM, EEPROM, CD-ROM, solid-state drive ("SSD"), or other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other media that can be used to store required program code tools in hardware in the form of computer-executable instructions, data or data structures and that can be accessed by a general-purpose or special-purpose computer.

[0545] Those skilled in the art will appreciate that the present invention can be practiced in a network computing environment having many types of computer system configurations, including personal computers, desktop computers, laptop computers, message processors, handheld devices, multiprocessor systems, microprocessor-based or programmable consumer electronics, network PCs, minicomputers, mainframe computers, mobile phones, PDAs, pagers, routers, switches, wearable devices, and the like. The present invention can also be implemented in a distributed system environment in which multiple computer systems (e.g., local and remote systems) linked by a network (via hardwired data links, wireless data links, or a combination of hardwired and wireless data links) perform tasks. In a distributed system environment, program modules can be located in local and / or remote memory storage devices.

[0546] XI. Implementation Plan

[0547] Implementation Scheme 1. An oligonucleotide having the following formula or a salt thereof:

[0548] in

[0549] B is a divalent nucleobase;

[0550] L 1 It is a divalent linker;

[0551] L 5 is a bivalent oligonucleotide linker comprising 4 to 40 nucleotides;

[0552] L 50 is a bond, -NH-, -O-, -S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

[0553] R 50 is the detectable portion;

[0554] R 30 Yes-OR 3A or -OP(NR 3B R 3C )-OR 3A ;

[0555] R 30A It is hydrogen, a 3' closed portion, or a monovalent oligonucleotide portion;

[0556] R 2 Is it hydrogen or -OR 2A ;

[0557] R 4 is hydrogen or unsubstituted methyl, or R 2 and R 4 The substituents are linked to form a substituted or unsubstituted heterocycloalkyl group;

[0558] R 1 and R 10Independently, it is hydrogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl;

[0559] R 6 、R 7 、R 8 and R 9 are independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3R A , -SO2NH2, -NHNH2, -ONH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCBr3, -OCF3, -OCI3, -OCH2Cl, -OCH2Br, -OCH2F, -OCHI, -OCHCl2, -OCHBr2, -OCHF2, -OCHI2, -SF5, -N3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

[0560] R 1 and R 6 capable of linking to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group;

[0561] R 8 and R 10 capable of being linked to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group; and

[0562] R 2A 、R 3A 、R 3B 、R 3C and R Aand -CHCl, -CBr, -CF, -CI, -CHCl, -CHBr, -CHF, -CHI, -CHCl, -CHBr, -CHF, -CHI, -CN, -OH, -NH, -COOH, -CONH, -OCCl, -OCF, -OCBr, -OCI, -OCHCl, -OCHBr, -OCHI, -OCHF, -OCHCl, -OCHBr, -OCHI, -OCHF, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0563] Implementation Scheme 2. The oligonucleotide according to Implementation Scheme 1 has the following formula:

[0564]

[0565] Implementation Scheme 3. The oligonucleotide according to Implementation Scheme 1 has the following formula:

[0566]

[0567] Implementation Scheme 4. The oligonucleotide according to Implementation Scheme 1 has the following formula:

[0568]

[0569] Implementation Scheme 5. The oligonucleotide according to Implementation Scheme 1 has the following formula:

[0570]

[0571] Implementation Scheme 6. An oligonucleotide according to any one of Implementation Schemes 1 to 5, wherein B is divalent cytosine or a derivative thereof, divalent guanine or a derivative thereof, divalent adenine or a derivative thereof, divalent thymine or a derivative thereof, divalent uracil or a derivative thereof, divalent hypoxanthine or a derivative thereof, divalent xanthine or a derivative thereof, divalent 7-methylguanine or a derivative thereof, divalent 5,6-dihydrouracil or a derivative thereof, divalent 5-methylcytosine or a derivative thereof, or divalent 5-hydroxymethylcytosine or a derivative thereof.

[0572] Embodiment 7. The oligonucleotide according to one of embodiments 1 to 5, wherein B is a divalent cytosine or a derivative thereof, a divalent guanine or a derivative thereof, a divalent adenine or a derivative thereof, a divalent thymine or a derivative thereof, or a divalent uracil or a derivative thereof.

[0573] Embodiment 8. The oligonucleotide of embodiment 1, which has the formula:

[0574]

[0575] Embodiment 9. The oligonucleotide of embodiment 1, which has the formula:

[0576]

[0577] Embodiment 10. The oligonucleotide of embodiment 1, which has the formula:

[0578]

[0579] Embodiment 11. The oligonucleotide of embodiment 1, which has the formula:

[0580]

[0581] Embodiment 12. The oligonucleotide according to one of embodiments 1 to 11, wherein L 5 It contains 11 to 30 nucleotides.

[0582] Embodiment 13. The oligonucleotide according to one of embodiments 1 to 11, wherein L 5 It contains 19 to 23 nucleotides.

[0583] Embodiment 14. The oligonucleotide according to one of embodiments 1 to 11, wherein L 5 Contains 4 to 14 nucleotides.

[0584] Embodiment 15. The oligonucleotide according to one of embodiments 1 to 11, wherein L 5 Contains 6 to 12 nucleotides.

[0585] Implementation Scheme 16. An oligonucleotide according to any one of Implementation Schemes 1 to 15, wherein the nucleotide is a DNA nucleotide.

[0586] Implementation Scheme 17. An oligonucleotide according to any one of Implementation Schemes 1 to 15, wherein the nucleotide is an RNA nucleotide.

[0587] Embodiment 18. The oligonucleotide according to one of embodiments 1 to 17, wherein L 1 It's L 101 -L 102 -L 103 -L 104 -L 105 ;and L 101 , L102 , L 103 , L 104 and L 105 Independently, it is a bond, -NH-, -O-, -S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene.

[0588] Implementation Scheme 19. The oligonucleotide according to Implementation Scheme 18, wherein L 101 It is -S(O)2-.

[0589] Implementation Scheme 20. The oligonucleotide according to any one of Implementation Schemes 18 to 19, wherein L 102 It is an unsubstituted 3- to 8-membered heterocyclic alkyl group.

[0590] Implementation Scheme 21. The oligonucleotide according to any one of Implementation Schemes 18 to 19, wherein L 102 It is an unsubstituted piperidinyl group.

[0591] Implementation Scheme 22. The oligonucleotide according to one of Implementation Schemes 18 to 19, wherein L 102 yes

[0592] Implementation Scheme 23. The oligonucleotide according to any one of Implementation Schemes 18 to 22, wherein L 103 It is -C(O)NH-.

[0593] Implementation Scheme 24. The oligonucleotide according to any one of Implementation Schemes 18 to 23, wherein L 104 is an unsubstituted C1-C 10 Alkylene, unsubstituted 2- to 6-membered heteroalkylene or unsubstituted phenylene.

[0594] Implementation Scheme 25. The oligonucleotide according to any one of Implementation Schemes 18 to 23, wherein L 104 is an unsubstituted n-hexamethylene group,

[0595] Implementation Scheme 26. The oligonucleotide according to any one of Implementation Schemes 18 to 25, wherein L 105 is an unsubstituted C1-C 10 Alkylene, substituted or unsubstituted 2 to 8-membered heteroalkylene or unsubstituted 5 to 10-membered heteroarylene.

[0596] Embodiment 27. The oligonucleotide according to one of embodiments 18 to 25, wherein L 105 yes

[0597] Implementation Scheme 28. An oligonucleotide according to any one of Implementation Schemes 1 to 17, wherein L 1 yes

[0598] Implementation Scheme 29. The oligonucleotide according to any one of Implementation Schemes 1 to 28, wherein L 50 is a substituted 2- to 10-membered heteroalkylene group.

[0599] Embodiment 30. The oligonucleotide according to one of embodiments 1 to 28, wherein L 50 yes

[0600] Implementation Scheme 31. The oligonucleotide according to any one of Implementation Schemes 1 to 30, wherein R 50 It is the fluorescent part.

[0601] Implementation Scheme 32. The oligonucleotide according to Implementation Scheme 31, wherein R 50 It is a monovalent form of FAM, a monovalent form of VIC, a monovalent form of ABY, a monovalent form of JUN, a monovalent form of AF647, a monovalent form of Cy5, a monovalent form of AF676, or a monovalent form of Cy5.5.

[0602] Embodiment 33. The oligonucleotide according to one of embodiments 1 to 32, wherein R 2 It is hydrogen or -OH.

[0603] Implementation Scheme 34. The oligonucleotide according to any one of Implementation Schemes 1 to 32, wherein R 2 It's hydrogen.

[0604] Embodiment 35. The oligonucleotide according to one of embodiments 1 to 34, wherein R 30 It is -OH.

[0605] Implementation Scheme 36. The oligonucleotide according to any one of Implementation Schemes 1 to 34, wherein R 30 yes

[0606] Implementation Scheme 37. An oligonucleotide according to any one of Implementation Schemes 1 to 34, wherein the 3′ blocking portion is a monovalent form of dideoxycytidine (3′ddC), a monovalent form of dideoxyadenosine (ddA), a 3′ reverse dT, a 3′ amino modifier, a monovalent form of QSY7, a monovalent form of QSY21, a monovalent form of QSY9, a monovalent form of BHQ1, a monovalent form of BHQ2, a monovalent form of BHQ3, a monovalent form of Dabcyl, a monovalent form of Dabsyl, a monovalent form of Eclipse, a monovalent form of BBQ-650, a monovalent form of Iowa Black RQ, or a monovalent form of Iowa Black FQ.

[0607] Implementation Scheme 38. A compound having the following formula or a salt thereof:

[0608] in

[0609] B is a divalent nucleobase;

[0610] L 1 It is a divalent linker;

[0611] R 2 Is it hydrogen or -OR 2A ;

[0612] R 3 Yes-OR 3A or -OP(NR 3B R 3C )-OR 3A ;

[0613] R 4 is hydrogen or unsubstituted methyl, or R 2 and R 4 The substituents are linked to form a substituted or unsubstituted heterocycloalkyl group;

[0614] R 5 Yes-OR 5A ;

[0615] R 1 and R 10Independently, it is hydrogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl;

[0616] R 6 、R 7 、R 8 and R 9 are independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3R A , -SO2NH2, -NHNH2, -ONH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCBr3, -OCF3, -OCI3, -OCH2Cl, -OCH2Br, -OCH2F, -OCHI, -OCHCl2, -OCHBr2, -OCHF2, -OCHI2, -SF5, -N3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

[0617] R 1 and R 6 capable of linking to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group;

[0618] R 8 and R 10 capable of being linked to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group; and

[0619] R 2A 、R 3A 、R 3B 、R 3C 、R 5A and R Aand -CHCl, -CBr, -CF, -CI, -CHCl, -CHBr, -CHF, -CHI, -CHCl, -CHBr, -CHF, -CHI, -CN, -OH, -NH, -COOH, -CONH, -OCCl, -OCF, -OCBr, -OCI, -OCHCl, -OCHBr, -OCHI, -OCHF, -OCHCl, -OCHBr, -OCHI, -OCHF, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0620] Implementation Scheme 39. The compound according to Implementation Scheme 38, the compound having the following formula:

[0621]

[0622] Implementation Scheme 40. The compound according to Implementation Scheme 38, the compound having the following formula:

[0623]

[0624] Implementation Scheme 41. The compound according to Implementation Scheme 38, the compound having the following formula:

[0625]

[0626] Implementation Scheme 42. The compound according to Implementation Scheme 38, the compound having the following formula:

[0627]

[0628] Embodiment 43. The compound according to one of Embodiments 38 to 42, wherein B is a divalent cytosine or a derivative thereof, a divalent guanine or a derivative thereof, a divalent adenine or a derivative thereof, a divalent thymine or a derivative thereof, a divalent uracil or a derivative thereof, a divalent hypoxanthine or a derivative thereof, a divalent xanthine or a derivative thereof, a divalent 7-methylguanine or a derivative thereof, a divalent 5,6-dihydrouracil or a derivative thereof, a divalent 5-methylcytosine or a derivative thereof, or a divalent 5-hydroxymethylcytosine or a derivative thereof.

[0629] Implementation Scheme 44. The compound according to any one of Implementation Schemes 38 to 42, wherein B is a divalent cytosine or a derivative thereof, a divalent guanine or a derivative thereof, a divalent adenine or a derivative thereof, a divalent thymine or a derivative thereof, or a divalent uracil or a derivative thereof.

[0630] Implementation Scheme 45. The compound according to Implementation Scheme 38, the compound having the following formula:

[0631]

[0632] Embodiment 46. The compound of embodiment 38, which has the following formula:

[0633]

[0634] Embodiment 47. The compound according to embodiment 38, which has the following formula:

[0635]

[0636] Implementation Scheme 48. The oligonucleotide according to Implementation Scheme 38 has the following formula:

[0637]

[0638] Implementation Scheme 49. The compound according to any one of Implementation Schemes 38 to 48, wherein L 1 It's L 101 -L 102 -L 103 -L 104 -L 105 ;and L 101 、L 102 、L 103 、L 104 and L 105 and -NH-, -O-, -S-, -S(O)-, -S(O)-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene.

[0639] Embodiment 50. The compound according to embodiment 49, wherein L 101 It is -S(O)2-.

[0640] Implementation Scheme 51. The compound according to any one of Implementation Schemes 49 to 50, wherein L 102 is an unsubstituted 3- to 8-membered heterocycloalkyl group.

[0641] Implementation Scheme 52. The compound according to any one of Implementation Schemes 49 to 50, wherein L 102 is an unsubstituted piperidinyl.

[0642] Implementation Scheme 53. The compound according to any one of Implementation Schemes 49 to 50, wherein L 102 yes

[0643] Implementation Scheme 54. The compound according to any one of Implementation Schemes 49 to 53, wherein L 103 It is -C(O)NH-.

[0644] Embodiment 55. The compound according to one of embodiments 49 to 54, wherein L 104 is an unsubstituted C1-C 10 an alkylene group, an unsubstituted 2- to 6-membered heteroalkylene group, or an unsubstituted phenylene group.

[0645] Embodiment 56. The compound according to one of embodiments 49 to 54, wherein L 104 It is unsubstituted positive hexylene,

[0646] Implementation Scheme 57. The compound according to any one of Implementation Schemes 49 to 56, wherein L 105 is an unsubstituted C1-C 10 Alkylene, substituted or unsubstituted 2 to 8-membered heteroalkylene or unsubstituted 5 to 10-membered heteroarylene.

[0647] Embodiment 58. The compound according to one of embodiments 49 to 56, wherein L 105 yes

[0648] Embodiment 59. The compound according to one of embodiments 38 to 48, wherein L 1 yes

[0649]

[0650]

[0651] Implementation Scheme 60. The compound according to any one of Implementation Schemes 38 to 40 and 43 to 59, wherein R 2 It is hydrogen or -OH.

[0652] Implementation Scheme 61. The compound according to any one of Implementation Schemes 38 to 40 and 43 to 59, wherein R 2 It's hydrogen.

[0653] Embodiment 62. The compound according to one of embodiments 38 to 61, wherein R 3 It is -OH.

[0654] Implementation Scheme 63. The compound according to any one of Implementation Schemes 38 to 61, wherein R 3 yes

[0655] Implementation Scheme 64. The compound according to any one of Implementation Schemes 38 to 63, wherein R 5A It is hydrogen or substituted C1-C6 alkyl.

[0656] Implementation Scheme 65. The compound according to any one of Implementation Schemes 38 to 63, wherein R 5A It is dimethoxytrityl.

[0657] Embodiment 66. The compound of embodiment 38, which has the following formula:

[0658]

[0659] Implementation Scheme 67. The compound according to Implementation Scheme 38, the compound having the following formula:

[0660]

[0661] Implementation Scheme 68. The compound according to Implementation Scheme 38, the compound having the following formula:

[0662]

[0663] Implementation Scheme 69. The compound according to Implementation Scheme 38, the compound having the following formula:

[0664]

[0665] Embodiment 70. A method of forming a double-stranded nucleic acid, the method comprising contacting a target oligonucleotide with a nucleic acid probe comprising a quencher moiety, thereby forming the double-stranded nucleic acid; wherein the nucleic acid probe has the formula:

[0666] (XIX) or a salt thereof, wherein

[0667] Q is a quencher moiety having the formula:

[0668]

[0669] B is a divalent nucleobase;

[0670] L 1 It is a divalent linker;

[0671] L 5 is a bivalent oligonucleotide linker comprising 4 to 40 nucleotides;

[0672] L 50 is a bond, -NH-, -O-, -S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

[0673] R 50 is the detectable portion;

[0674] R 30 Yes-OR 30A ;

[0675] R 30A is a monovalent oligonucleotide moiety;

[0676] R 2 Is it hydrogen or -OR 2A ;

[0677] R 4 is hydrogen or unsubstituted methyl, or R 2 and R 4 The substituents are linked to form a substituted or unsubstituted heterocycloalkyl group;

[0678] R 1 and R 10 is independently hydrogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCHI, -OCH2F, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl;

[0679] R 6 、R 7 、R 8 and R 9 are independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3R A, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCBr3, -OCF3, -OCI3, -OCH2Cl, -OCH2Br, -OCH2F, -OCHI, -OCHCl2, -OCHBr2, -OCHF2, -OCHI2, -SF5, -N3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

[0680] R 1 and R 6 capable of linking to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group;

[0681] R 8 and R 10 capable of being linked to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group; and

[0682] R 2A and R A and -CHCl, -CBr, -CF, -CI, -CHCl, -CHBr, -CHF, -CHI, -CHCl, -CHBr, -CHF, -CHI, -CN, -OH, -NH, -COOH, -CONH, -OCCl, -OCF, -OCBr, -OCI, -OCHCl, -OCHBr, -OCHI, -OCHF, -OCHCl, -OCHBr, -OCHI, -OCHF, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0683] Embodiment 71. The method according to embodiment 70, wherein L 5 Contains 11 to 30 nucleotides.

[0684] Embodiment 72. The method according to embodiment 70, wherein L 5 It contains 19 to 23 nucleotides.

[0685] Embodiment 73. The method according to embodiment 70, wherein L 5 Contains 4 to 14 nucleotides.

[0686] Embodiment 74. The method according to embodiment 70, wherein L 5It contains 6 to 12 nucleotides.

[0687] Embodiment 75. The method according to one of embodiments 70 to 74, wherein the nucleotide is a DNA nucleotide.

[0688] Implementation Scheme 76. The method according to one of Implementation Schemes 70 to 74, wherein the nucleotide is an RNA nucleotide.

[0689] Implementation Scheme 77. The method according to one of Implementation Schemes 70 to 76, the method further comprising contacting the double-stranded nucleic acid with a polymerase having exonuclease activity, thereby cleaving the quencher portion from the detectable portion.

[0690] Embodiment 78. The method of embodiment 77, further comprising detecting a signal produced by the detectable portion.

[0691] Embodiment 79. The method of one of embodiments 70 to 78, wherein the detectable moiety is a fluorescent moiety.

[0692] Implementation Scheme 80. The method according to one of Implementation Schemes 78 to 79, further comprising amplifying the target oligonucleotide using PCR.

[0693] Implementation Scheme 81. The method according to Implementation Scheme 80, wherein the PCR is quantitative PCR.

[0694] Embodiment 82. A method according to embodiment 80, wherein the PCR is digital PCR.

[0695] Embodiment 83. A method for detecting nucleic acid in a sample, the method comprising:

[0696] (i) A reaction mixture is provided, the reaction mixture comprising:

[0697] At least a portion of the sample,

[0698] a first probe detectably labeled with a first label configured to produce a first fluorescent signal,

[0699] A second probe, detectably labeled with a second marker configured to generate a second fluorescent signal.

[0700] The first probe and the second probe have different sequences.

[0701] Wherein the first label and the second label are identical and / or produce substantially the same fluorescence; and

[0702] The second probe has the following formula:

[0703] (XIX) or a salt thereof, wherein

[0704] Q is a quencher moiety having the formula:

[0705]

[0706] B is a divalent nucleobase;

[0707] L 1 It is a divalent linker;

[0708] L 5 is a bivalent oligonucleotide linker comprising 4 to 40 nucleotides;

[0709] L 50 is a bond, -NH-, -O-, -S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

[0710] R 50 It is the second mark;

[0711] R 30 Yes-OR 30A ;

[0712] R 30A is a monovalent oligonucleotide moiety;

[0713] R 2 Is it hydrogen or -OR 2A ;

[0714] R 4 is hydrogen or unsubstituted methyl, or R 2 and R 4 The substituents are linked to form a substituted or unsubstituted heterocycloalkyl group;

[0715] R 1 and R 10is independently hydrogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCHI, -OCH2F, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl;

[0716] R 6 、R 7 、R 8 and R 9 are independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3R A , -SO2NH2, -NHNH2, -ONH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCBr3, -OCF3, -OCI3, -OCH2Cl, -OCH2Br, -OCH2F, -OCHI, -OCHCl2, -OCHBr2, -OCHF2, -OCHI2, -SF5, -N3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 1 and R 6 capable of linking to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group;

[0717] R 8 and R 10 capable of being linked to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group; and

[0718] R 2A and R AIndependently, it is hydrogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

[0719] (ii) subjecting the reaction mixture to an amplification process comprising a first set of reaction conditions and a second set of reaction conditions, the first set of reaction conditions being different from the second set of reaction conditions; and

[0720] (iii) determining the presence of the first nucleic acid target and / or the second nucleic acid target in the sample by the following steps:

[0721] measuring a fluorescent signal during the first set of reaction conditions, the fluorescent signal during the first set of reaction conditions being correlated with a specific interaction, or lack of interaction, between the first probe and the first nucleic acid target,

[0722] measuring a fluorescent signal during the second set of reaction conditions, the fluorescent signal during the second set of reaction conditions being correlated with the specific interaction, or lack thereof, between the first probe and the first nucleic acid target and with the specific interaction, or lack thereof, between the second probe and the second nucleic acid target, and

[0723] The presence and / or amount of the first nucleic acid target and the second nucleic acid target are estimated.

[0724] Embodiment 84. A method for detecting nucleic acid in a sample, the method comprising:

[0725] (i) A reaction mixture is provided, the reaction mixture comprising:

[0726] At least a portion of the sample,

[0727] a first probe detectably labeled with a first label configured to generate a first fluorescent signal indicative of the presence or absence of a first nucleic acid target,

[0728] a second probe detectably labeled with a second label configured to produce a second fluorescent signal indicative of the presence or absence of a second nucleic acid target,

[0729] wherein the first probe and the second probe have different sequences,

[0730] Wherein the first label and the second label are identical and / or produce substantially the same fluorescence; and

[0731] The second probe has the following formula:

[0732] (XIX) or its salt, wherein

[0733] Q is the quencher component with the following formula:

[0734]

[0735] B is a divalent nucleobase;

[0736] L 1 It is a bivalent connector;

[0737] L 5 is a bivalent oligonucleotide linker comprising 4 to 40 nucleotides;

[0738] L 50 is a bond, -NH-, -O-, -S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

[0739] R 50 It is the second mark;

[0740] R 30 Yes - OR 30A ;

[0741] R 30A It is the monovalent oligonucleotide moiety;

[0742] R 2 Is it hydrogen or -OR 2A ;

[0743] R 4 It is hydrogen or an unsubstituted methyl group, or R 2 and R 4 The substituents are linked to form a substituted or unsubstituted hetero...

Claims

1. An oligonucleotide having the formula: in B is a divalent nucleobase; L 1 It is a divalent linker; L 5 is a divalent oligonucleotide linker comprising 4 to 40 nucleotides; L 50 is a bond, -NH-, -O-, -S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; R 50 is the detectable portion; R 30 is - OR 3A or - O - P(NR 3B R 3C ) - OR 3A ; R 30A is hydrogen, a 3' blocking moiety, or a monovalent oligonucleotide moiety; R 2 is hydrogen or -OR 2A ; R 4 is hydrogen or unsubstituted methyl, or R 2 and R 4 The substituents are linked to form a substituted or unsubstituted heterocycloalkyl group; R 1 and R 10 are independently hydrogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R 6 , R 7 , R 8 and R 9 are independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3R A , -SO2NH2, -NHNH2, -ONH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCBr3, -OCF3, -OCI3, -OCH2Cl, -OCH2Br, -OCH2F, -OCHI, -OCHCl2, -OCHBr2, -OCHF2, -OCHI2, -SF5, -N3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 1 and R 6 Can be linked to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group; R 8 and R 10 capable of being linked to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group; and R 2A , R 3A , R 3B , R 3C and R A and -CHCl, -CHBr, -CHF, -CHI, -CHCl, -CHBr, -CHF, -CHI, -CN, -OH, -NH, -COOH, -CONH, -OCCl, -OCF, -OCBr, -OCI, -OCHCl, -OCHBr, -OCHI, -OCHF, -OCHCl, -OCHBr, -OCHI, -OCHF, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

2. The oligonucleotide according to claim 1, which has the formula:

3. The oligonucleotide according to claim 1, wherein the oligonucleotide has the following formula:

4. The oligonucleotide according to claim 1, wherein the oligonucleotide has the following formula:

5. The oligonucleotide according to claim 1, wherein the oligonucleotide has the following formula:

6. The oligonucleotide according to claim 1, wherein B is a divalent cytosine or a derivative thereof, a divalent guanine or a derivative thereof, a divalent adenine or a derivative thereof, a divalent thymine or a derivative thereof, a divalent uracil or a derivative thereof, a divalent hypoxanthine or a derivative thereof, a divalent xanthine or a derivative thereof, a divalent 7-methylguanine or a derivative thereof, a divalent 5,6-dihydrouracil or a derivative thereof, a divalent 5-methylcytosine or a derivative thereof, or a divalent 5-hydroxymethylcytosine or a derivative thereof.

7. The oligonucleotide according to claim 1, wherein B is a divalent cytosine or a derivative thereof, a divalent guanine or a derivative thereof, a divalent adenine or a derivative thereof, a divalent thymine or a derivative thereof, or a divalent uracil or a derivative thereof.

8. The oligonucleotide according to claim 1, wherein the oligonucleotide has the formula:

9. The oligonucleotide according to claim 1, wherein the oligonucleotide has the formula:

10. The oligonucleotide of claim 1, wherein the oligonucleotide has the formula:

11. The oligonucleotide according to claim 1, which has the formula:

12. The oligonucleotide according to claim 1, wherein L 5 Contains 11 to 30 nucleotides.

13. The oligonucleotide according to claim 1, wherein L 5 Contains 19 to 23 nucleotides.

14. The oligonucleotide according to claim 1, wherein L 5 Contains 4 to 14 nucleotides.

15. The oligonucleotide according to claim 1, wherein L 5 Contains 6 to 12 nucleotides.

16. The oligonucleotide of claim 1, wherein the nucleotides are DNA nucleotides.

17. The oligonucleotide of claim 1, wherein the nucleotides are RNA nucleotides.

18. The oligonucleotide according to claim 1, wherein L 1 YesL 101 -L 102 -L 103 -L 104 -L 105 ;and L 101 , L 102 , L 103 , L 104 and L 105 are independently a bond, -NH-, -O-, -S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene.

19. The oligonucleotide according to claim 18, wherein L 101 It is -S(O)2-.

20. The oligonucleotide according to claim 18, wherein L 102 is an unsubstituted 3- to 8-membered heterocycloalkyl group.

21. The oligonucleotide according to claim 18, wherein L 102 is an unsubstituted piperidinyl.

22. The oligonucleotide according to claim 18, wherein L 102 yes 23. The oligonucleotide according to claim 18, wherein L 103 It is –C(O)NH-.

24. The oligonucleotide according to claim 18, wherein L 104 is an unsubstituted C1-C 10 alkylene, unsubstituted 2- to 6-membered heteroalkylene or unsubstituted phenylene.

25. The oligonucleotide according to claim 18, wherein L 104 is an unsubstituted n-hexylene, 26. The oligonucleotide according to claim 18, wherein L 105 is an unsubstituted C1-C 10 alkylene, substituted or unsubstituted 2- to 8-membered heteroalkylene, or unsubstituted 5- to 10-membered heteroarylene.

27. The oligonucleotide according to claim 18, wherein L 105 yes 28. The oligonucleotide according to claim 1, wherein L 1 yes 29. The oligonucleotide according to claim 1, wherein L 50 is a substituted 2- to 10-membered heteroalkylene group.

30. The oligonucleotide according to claim 1, wherein L 50 yes 31. The oligonucleotide according to claim 1, wherein R 50 It is the fluorescent part.

32. The oligonucleotide according to claim 31, wherein R 50 It is a monovalent form of FAM, a monovalent form of VIC, a monovalent form of ABY, a monovalent form of JUN, a monovalent form of AF647, a monovalent form of Cy5, a monovalent form of AF676, or a monovalent form of Cy5.

5.

33. The oligonucleotide according to claim 1, wherein R 2 It is hydrogen or -OH.

34. The oligonucleotide according to claim 1, wherein R 2 It's hydrogen.

35. according to the oligonucleotide of one of claims 1 to 34, wherein R 30 It is -OH.

36. according to the oligonucleotide of one of claims 1 to 34, wherein R 30 yes 37. The oligonucleotide of one of claims 1 to 34, wherein the 3′ blocking moiety is a monovalent form of dideoxycytidine (3′ddC), a monovalent form of dideoxyadenosine (ddA), 3′ reverse dT, a 3′ amino modifier, a monovalent form of QSY7, a monovalent form of QSY21, a monovalent form of QSY9, a monovalent form of BHQ 1, a monovalent form of BHQ2, a monovalent form of BHQ3, a monovalent form of Dabcyl, a monovalent form of Dabsyl, a monovalent form of Eclipse, a monovalent form of BBQ-650, a monovalent form of Iowa Black RQ, Iowa Black FQ unit price format, 38. A compound having the formula: in B is a divalent nucleobase; L 1 It is a divalent linker; R 2 is hydrogen or -OR 2A ; R 3 is - OR 3A or - O - P(NR 3B R 3C ) - OR 3A ; R 4 is hydrogen or unsubstituted methyl, or R 2 and R 4 The substituents are linked to form a substituted or unsubstituted heterocycloalkyl group; R 5 is - OR 5A ; R 1 and R 10 are independently hydrogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R 6 , R 7 , R 8 and R 9 are independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3R A , -SO2NH2, -NHNH2, -ONH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCBr3, -OCF3, -OCI3, -OCH2Cl, -OCH2Br, -OCH2F, -OCHI, -OCHCl2, -OCHBr2, -OCHF2, -OCHI2, -SF5, -N3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 1 and R 6 Can be linked to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group; R 8 and R 10 capable of being linked to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group; and R 2A , R 3A , R 3B , R 3C , R 5A and R A and -CHCl, -CHBr, -CHF, -CHI, -CHCl, -CHBr, -CHF, -CHI, -CN, -OH, -NH, -COOH, -CONH, -OCCl, -OCF, -OCBr, -OCI, -OCHCl, -OCHBr, -OCHI, -OCHF, -OCHCl, -OCHBr, -OCHI, -OCHF, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

39. The compound of claim 38, which has the formula:

40. The compound of claim 38, which has the formula:

41. The compound of claim 38, which has the formula:

42. The compound of claim 38, which has the formula:

43. The compound according to claim 38, wherein B is divalent cytosine or a derivative thereof, divalent guanine or a derivative thereof, divalent adenine or a derivative thereof, divalent thymine or a derivative thereof, divalent uracil or a derivative thereof, divalent hypoxanthine or a derivative thereof, divalent divalent xanthine or a derivative thereof, divalent 7-methylguanine or a derivative thereof, divalent 5,6-dihydrouracil or a derivative thereof, divalent 5-methylcytosine or a derivative thereof, or divalent 5-hydroxymethylcytosine or a derivative thereof.

44. The compound of claim 38, wherein B is divalent cytosine or a derivative thereof, divalent guanine or a derivative thereof, divalent adenine or a derivative thereof, divalent thymine or a derivative thereof, or divalent uracil or a derivative thereof.

45. The compound of claim 38, which has the formula:

46. ​​The compound of claim 38, which has the formula:

47. The compound of claim 38, which has the formula:

48. The compound of claim 38, which has the formula:

49. The compound according to claim 38, wherein L 1 YesL 101 -L 102 -L 103 -L 104 -L 105 ; and L 101 , L 102 , L 103 , L 104 and L 105 are independently a bond, -NH-, -O-, -S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene.

50. The compound according to claim 49, wherein L 101 It is -S(O)2-.

51. The compound according to claim 49, wherein L 102 is an unsubstituted 3- to 8-membered heterocycloalkyl group.

52. The compound according to claim 49, wherein L 102 is an unsubstituted piperidinyl.

53. The compound according to claim 49, wherein L 102 yes 54. The compound according to claim 49, wherein L 103 It is -C(O)NH-.

55. The compound according to claim 49, wherein L 104 is an unsubstituted C1-C 10 alkylene, unsubstituted 2- to 6-membered heteroalkylene or unsubstituted phenylene.

56. The compound according to claim 49, wherein L 104 is an unsubstituted n-hexylene, 57. The compound according to claim 49, wherein L 105 is an unsubstituted C1-C 10 alkylene, substituted or unsubstituted 2- to 8-membered heteroalkylene, or unsubstituted 5- to 10-membered heteroarylene.

58. The compound according to claim 49, wherein L 105 yes 59. The compound according to claim 38, wherein L 1 yes 60. The compound according to claim 38, wherein R 2 It is hydrogen or -OH.

61. The compound according to claim 38, wherein R 2 It's hydrogen.

62. The compound according to claim 38, wherein R 3 It is -OH.

63. The compound according to claim 38, wherein R 3 yes 64. according to the compound described in one of claim 38 to 63, wherein R 5A It is hydrogen or substituted C1-C6 alkyl.

65. according to the compound described in one of claim 38 to 63, wherein R 5A It is dimethoxytrityl.

66. The compound of claim 38, which has the formula:

67. The compound of claim 38, which has the formula:

68. The compound of claim 38, which has the formula:

69. The compound of claim 38, which has the formula:

70. A method of forming a double-stranded nucleic acid, the method comprising contacting a target oligonucleotide with a nucleic acid probe comprising a quencher moiety, thereby forming the double-stranded nucleic acid; wherein the nucleic acid probe has the formula: or a salt thereof, wherein Q is the quencher moiety having the formula: B is a divalent nucleobase; L 1 It is a divalent linker; L 5 is a divalent oligonucleotide linker comprising 4 to 40 nucleotides; L 50 is a bond, -NH-, -O-, -S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; R 50 is the detectable portion; R 30 is - OR 30A ; R 30A is a monovalent oligonucleotide moiety; R 2 is hydrogen or -OR 2A ; R 4 is hydrogen or unsubstituted methyl, or R 2 and R 4 The substituents are linked to form a substituted or unsubstituted heterocycloalkyl group; R 1 and R 10 are independently hydrogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R 6 , R 7 , R 8 and R 9 are independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3R A , -SO2NH2, -NHNH2, -ONH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCBr3, -OCF3, -OCI3, -OCH2Cl, -OCH2Br, -OCH2F, -OCHI, -OCHCl2, -OCHBr2, -OCHF2, -OCHI2, -SF5, -N3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 1 and R 6 Can be linked to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group; R 8 and R 10 capable of being linked to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group; and R 2A and R A and -CHCl, -CHBr, -CHF, -CHI, -CHCl, -CHBr, -CHF, -CHI, -CN, -OH, -NH, -COOH, -CONH, -OCCl, -OCF, -OCBr, -OCI, -OCHCl, -OCHBr, -OCHI, -OCHF, -OCHCl, -OCHBr, -OCHI, -OCHF, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

71. The method of claim 70, wherein L 5 Contains 11 to 30 nucleotides.

72. The method of claim 70, wherein L 5 Contains 19 to 23 nucleotides.

73. The method of claim 70, wherein L 5 Contains 4 to 14 nucleotides.

74. The method of claim 70, wherein L 5 Contains 6 to 12 nucleotides.

75. The method of claim 70, wherein the nucleotides are DNA nucleotides.

76. The method of claim 70, wherein the nucleotides are RNA nucleotides.

77. The method of claim 70, further comprising contacting the double-stranded nucleic acid with a polymerase having exonuclease activity, thereby cleaving the quencher moiety from the detectable moiety.

78. The method of claim 77, further comprising detecting a signal produced by the detectable moiety.

79. The method of one of claims 70 to 78, wherein the detectable moiety is a fluorescent moiety.

80. The method of claim 78, further comprising amplifying the target oligonucleotide using PCR.

81. The method of claim 80, wherein the PCR is quantitative PCR.

82. The method of claim 80, wherein the PCR is digital PCR.

83. A method for detecting nucleic acid in a sample, the method comprising: (i) providing a reaction mixture, the reaction mixture comprising: at least a portion of the sample, a first probe detectably labeled with a first label configured to produce a first fluorescent signal, a second probe detectably labeled with a second label configured to produce a second fluorescent signal, wherein the first probe and the second probe have different sequences, wherein the first label and the second label are the same and / or produce substantially the same fluorescence; and Wherein the second probe has the following formula: or a salt thereof, wherein Q is a quencher moiety having the formula: B is a divalent nucleobase; L 1 It is a divalent linker; L 5 is a divalent oligonucleotide linker comprising 4 to 40 nucleotides; L 50 is a bond, -NH-, -O-, -S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; R 50 It is the second mark; R 30 is - OR 30A ; R 30A is a monovalent oligonucleotide moiety; R 2 is hydrogen or -OR 2A ; R 4 is hydrogen or unsubstituted methyl, or R 2 and R 4 The substituents are linked to form a substituted or unsubstituted heterocycloalkyl group; R 1 and R 10 are independently hydrogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R 6 , R 7 , R 8 and R 9 are independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3R A , -SO2NH2, -NHNH2, -ONH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCBr3, -OCF3, -OCI3, -OCH2Cl, -OCH2Br, -OCH2F, -OCH2I, -OCHCl2, -OCHB r2 , -OCHF2, -OCHI2, -SF5, -N3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 1 and R 6 Can be linked to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group; R 8 and R 10 capable of being linked to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group; and R 2A and R A are independently hydrogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCHI, -OCH2F, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; (ii) subjecting the reaction mixture to an amplification process comprising a first set of reaction conditions and a second set of reaction conditions, the first set of reaction conditions being different from the second set of reaction conditions; as well as (iii) determining the presence of the first nucleic acid target and / or the second nucleic acid target in the sample by the following steps: measuring a fluorescent signal during the first set of reaction conditions, the fluorescent signal during the first set of reaction conditions being associated with a specific interaction, or lack of interaction, between the first probe and the first nucleic acid target, measuring a fluorescent signal during the second set of reaction conditions, the fluorescent signal during the second set of reaction conditions being associated with a specific interaction, or lack thereof, between the first probe and the first nucleic acid target and being associated with a specific interaction, or lack thereof, between the second probe and the second nucleic acid target, and The presence and / or amount of the first nucleic acid target and the second nucleic acid target is estimated.

84. A method for detecting nucleic acid in a sample, the method comprising: (i) providing a reaction mixture, the reaction mixture comprising: at least a portion of the sample, a first probe detectably labeled with a first label configured to generate a first fluorescent signal indicative of the presence or absence of a first nucleic acid target, a second probe detectably labeled with a second label configured to produce a second fluorescent signal indicative of the presence or absence of a second nucleic acid target, wherein the first probe and the second probe have different sequences, wherein the first label and the second label are the same and / or produce substantially the same fluorescence; and Wherein the second probe has the following formula: or a salt thereof, wherein Q is a quencher moiety having the formula: B is a divalent nucleobase; L 1 It is a divalent linker; L 5 is a divalent oligonucleotide linker comprising 4 to 40 nucleotides; L 50 is a bond, -NH-, -O-, -S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; R 50 It is the second mark; R 30 is - OR 30A ; R 30A is a monovalent oligonucleotide moiety; R 2 is hydrogen or -OR 2A ; R 4 is hydrogen or unsubstituted methyl, or R 2 and R 4 The substituents are linked to form a substituted or unsubstituted heterocycloalkyl group; R 1 and R 10 are independently hydrogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R 6 , R 7 , R 8 and R 9 are independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3R A , -SO2NH2, -NHNH2, -ONH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCBr3, -OCF3, -OCI3, -OCH2Cl, -OCH2Br, -OCH2F, -OCH2I, -OCHCl2, -OCHB r2 , -OCHF2, -OCHI2, -SF5, -N3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 1 and R 6 capable of being linked to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group; and R 8 and R 10 capable of being linked to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group; and R 2A and R A are independently hydrogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCHI, -OCH2F, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; (ii) subjecting the reaction mixture to an amplification process comprising a first set of reaction conditions and a second set of reaction conditions, the first set of reaction conditions being different from the second set of reaction conditions; as well as (iii) determining any presence and / or amount of the first nucleic acid target and / or the second nucleic acid target in the reaction mixture by the following steps: measuring a first total fluorescent signal during the first set of reaction conditions, the first total fluorescent signal including any first fluorescent signal, if present, and any second fluorescent signal, if present, measuring a second total fluorescent signal during the second set of reaction conditions, the second total fluorescent signal including any first fluorescent signal, if present, and any second fluorescent signal, if present, and The first fluorescent signal and / or the second fluorescent signal are estimated based on the first total fluorescent signal and the second total fluorescent signal.

85. The method according to one of claims 83 to 84, wherein L 5 Contains 11 to 30 nucleotides.

86. The method according to one of claims 83 to 84, wherein L 5 Contains 19 to 23 nucleotides.

87. The method according to one of claims 83 to 84, wherein L 5 Contains 4 to 14 nucleotides.

88. The method according to one of claims 83 to 84, wherein L 5 Contains 6 to 12 nucleotides.

89. A method according to one of claims 83 to 84, wherein the nucleotides are DNA nucleotides.

90. A method according to one of claims 83 to 84, wherein the nucleotides are RNA nucleotides.

91. The method of one of claims 83 to 84, wherein both the first probe and the second probe are excited at the same wavelength and / or both the first probe and the second probe are excited during detection of their respective first and second fluorescent signals.

92. The method according to one of claims 83 to 84, wherein: Measuring the fluorescent signal during the first set of reaction conditions includes measuring a combined signal including the first fluorescent signal and the second fluorescent signal during the first set of reaction conditions to obtain a first total fluorescent signal; Measuring the fluorescent signal during the second set of reaction conditions includes measuring a combined signal including the first fluorescent signal and the second fluorescent signal during the second set of reaction conditions to obtain a second total fluorescent signal; and Estimating the presence and / or amount of the first nucleic acid target and the second nucleic acid target includes estimating the first fluorescent signal and / or the second fluorescent signal based on the first total fluorescent signal and the second total fluorescent signal.

93. The method of claim 92, wherein the degree of difference in the second fluorescent signal between the first set of reaction conditions and the second set of reaction conditions is greater than the degree of difference in the first fluorescent signal between the first set of reaction conditions and the second set of reaction conditions.

94. The method of claim 92, wherein The first total fluorescence value includes (i) fluorescence from a first label that is free and unquenched in the reaction mixture and is emitted due to cleavage of the first label after hybridization of the first probe to the first amplicon, and (ii) background fluorescence of the second label, and The second total fluorescence value is based on (i) fluorescence from the first label, which is free and unquenched in the reaction mixture and is emitted due to cleavage of the first label after hybridization of the first probe to the first amplicon, and (ii) fluorescence from the second label, which is above the background fluorescence of the second label and is emitted due to hybridization of the second probe to the second amplicon.

95. The method of claim 92, further comprising: calculating the amount of the first nucleic acid target based on the first fluorescent signal; as well as The amount of the second nucleic acid target is calculated based on the second fluorescent signal.

96. The method of claim 92, wherein when the first nucleic acid target is present in the reaction mixture, the first fluorescent signal is above background levels during both the first set of reaction conditions and the second set of reaction conditions.

97. The method of claim 92, wherein the second fluorescent signal is above background levels during the second set of reaction conditions but is not above background levels during the first set of reaction conditions when the second nucleic acid target is present in the reaction mixture.

98. The method of claim 93, wherein the first set of reaction conditions comprises a first measurement temperature at which the first fluorescent signal is measured, and the second set of reaction conditions comprises a different second measurement temperature at which the second fluorescent signal is measured.

99. The method of claim 98, wherein the first measured temperature and the second measured temperature differ by at least about 10°C or more, about 15°C or more, about 20°C or more, about 25°C or more, or about 30°C or more.

100. The method of claim 98, wherein at least one of the first measured temperature or the second measured temperature is a denaturation temperature at which the DNA in the reaction mixture denatures, such as about 90°C or above.

101. The method of one of claims 83 to 84, wherein the reaction mixture is subjected to multiple amplification cycles, each cycle comprising the first set of reaction conditions and the second set of reaction conditions.

102. The method of one of claims 83 to 84, wherein the amplification process comprises thermal cycling.

103. The method of claim 102, wherein the first set of reaction conditions comprises a denaturation step at a first temperature of the thermal cycle.

104. The method of claim 103, wherein the second set of reaction conditions comprises an annealing and / or extension step ("annealing / extension step") at a second temperature of the thermal cycle, the second temperature being lower than the first temperature.

105. The method of one of claims 83 to 84, wherein the first probe is a cleavable probe.

106. The method of claim 105, wherein the first fluorescent signal increases when the cleavable probe is cleaved during the annealing / extension step.

107. A method according to claim 105, wherein the first probe comprises a fluorophore and a quencher, and wherein the first probe is configured such that fluorescence from the fluorophore is quenched by the quencher until the probe is cleaved during the annealing / extension phase of the amplification process.

108. The method of claim 107, wherein the first probe is a TaqMan probe.

109. The method of one of claims 83 to 84, wherein the second probe is a non-cleavable probe.

110. The method of claim 109, wherein when the second probe is single-stranded, L 5 Forming a stem-loop structure.

111. The method of claim 109, wherein Q and R 50 are spaced apart from each other so that when the second probe is single-stranded, R 50 is quenched, and when the second probe is incorporated into the double-stranded amplicon, the R 50 Not quenched.

112. The method of claim 111, wherein Q and R 50 Both are arranged at or near the stem-loop portion of the second probe.

113. A method according to one of claims 83 to 84, wherein the reaction mixture further comprises: a first primer pair complementary to a first nucleic acid target of the nucleic acid or its complement, the first nucleic acid target being configured to produce a first amplicon with which the first probe can hybridize; and a second primer pair complementary to a second nucleic acid target of the nucleic acid or its complement, the second nucleic acid target being configured to produce a second amplicon with which the second probe can hybridize.

114. The method of claim 113, wherein the second primer pair comprises a primer having a tail.

115. The method of claim 114, wherein the tail forms the 5' end of the primer having the tail.

116. The method of claim 114, wherein the second probe is capable of hybridizing to the tail or its complement.

117. The method of claim 108, wherein the amplification process utilizes a series of thermal cycling steps comprising at least three different target temperatures.

118. The method of claim 117, wherein the amplification process comprises a denaturation temperature and a plurality of different annealing / extension temperatures that vary throughout the amplification process.

119. The method of claim 118, wherein a first series of denaturation steps and annealing / extension steps are performed at a first annealing / extension temperature, and wherein a second series of denaturation steps and annealing / extension steps are performed at a second, different annealing / extension temperature.

120. The method of claim 119, wherein the first annealing / extension temperature is higher than the second annealing / extension temperature.

121. The method of claim 119, wherein the first series of denaturation steps and annealing / extension steps is cycled more times than the second series of denaturation steps and annealing / extension steps.

122. The method of claim 119, wherein the amplification process further comprises a third series of denaturation steps and annealing / extension steps performed using a third annealing / extension temperature.

123. The method of claim 122, wherein the third annealing / extension temperature is the same as the first annealing / extension temperature.

124. The method of claim 122, wherein the third series of denaturation steps and annealing / extension steps is cycled more times than the first series of denaturation steps and annealing / extension steps.

125. The method of claim 119, wherein the denaturation temperature is the same for each series of denaturation steps and annealing / extension steps.

126. The method of claim 123, wherein the second primer pair comprises a tailed primer and a tailless primer, and wherein the tailed primer and the tailless primer are provided at different concentrations.

127. The method of claim 126, wherein the tailless primer is provided at a higher concentration than the tailed primer.

128. The method of claim 127, wherein the tailless primer is provided at a concentration of about 2X to about 30X the concentration of the tailed primer, or at about 5X to about 25X the concentration of the tailed primer, or at a concentration of about 10X to about 20X the concentration of the tailed primer.

129. The method of claim 126, wherein the second probe is provided at a concentration that is different from the concentration of the tailed primer and the concentration of the tailless primer.

130. The method of claim 129, wherein the second probe is provided at a concentration higher than the concentration of the tailed primer.

131. The method of claim 129, wherein the second probe is provided at a concentration lower than the concentration of the tailless primer.

132. The method of claim 129, wherein the second probe is provided at a concentration of about 2X to about 10X the concentration of the tailed primer, or about 3X to about 7.5X the concentration of the tailed primer.

133. The method of one of claims 83 to 84, wherein the melting temperature (T m ) and the T of the second probe m Within about 8°C, or about 6°C, or about 4°C, or about 2°C of each other.

134. The method of one of claims 83 to 84, wherein the amplification process cycles the amplification process between two target temperatures for multiple cycles.

135. A method according to claim 134, wherein the amplification process cycles between two target temperatures for at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the cycles of the amplification process.

136. A method according to one of claims 83 to 84, wherein the reaction mixture is added to or formed within multiple reaction volumes, and wherein the amplification process is a digital PCR (dPCR) process.

137. A method according to claim 136, wherein measuring the fluorescence signal during the first set of reaction conditions includes a first endpoint measurement under the first set of reaction conditions, and wherein measuring the fluorescence signal during the second set of reaction conditions includes a second endpoint measurement under the second set of reaction conditions.

138. The method of claim 137, wherein estimating the presence and / or amount of the first nucleic acid target and the second nucleic acid target comprises: classifying the plurality of reaction volumes according to the measured signal at the first endpoint metric and according to the measured signal at the second endpoint metric; as well as Based on the classification, counts of reaction volumes in which the first probe shows activity and counts of reaction volumes in which the second probe shows activity are determined.

139. A method for detecting nucleic acid in a sample, the method comprising: A reaction mixture is provided, the reaction mixture comprising: a primer pair that is complementary to the nucleic acid target or its complement for producing an amplicon, and a non-cleavable probe configured to hybridize to the amplicon, the probe comprising a detectable label configured to provide a fluorescent signal corresponding to the amount of amplicon produced, subjecting the reaction mixture to an amplification process to produce the amplicon, wherein the label produces fluorescence during the amplification process without cleaving the probe, and wherein the amplification process utilizes a series of thermal cycling steps including at least three different target temperatures; as well as measuring the fluorescent signal from the probe; Wherein the probe has the formula: or a salt thereof, wherein Q is a quencher moiety having the formula: B is a divalent nucleobase; L 1 It is a divalent linker; L 5 is a divalent oligonucleotide linker comprising 4 to 40 nucleotides; L 50 is a bond, -NH-, -O-, -S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; R 50 is a detectable marker; R 30 is - OR 30A ; R 30A is a monovalent oligonucleotide moiety; R 2 is hydrogen or -OR 2A ; R 4 is hydrogen or unsubstituted methyl, or R 2 and R 4 The substituents are linked to form a substituted or unsubstituted heterocycloalkyl group; R 1 and R 10 are independently hydrogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R 6 , R 7 , R 8 and R 9 are independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3R A , -SO2NH2, -NHNH2, -ONH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCBr3, -OCF3, -OCI3, -OCH2Cl, -OCH2Br, -OCH2F, -OCHI, -OCHCl2, -OCHBr2, -OCHF2, -OCHI2, -SF5, -N3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 1 and R 6 Can be linked to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group; R 8 and R 10 capable of being linked to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group; and R 2A and R A and -CHCl, -CHBr, -CHF, -CHI, -CHCl, -CHBr, -CHF, -CHI, -CN, -OH, -NH, -COOH, -CONH, -OCCl, -OCF, -OCBr, -OCI, -OCHCl, -OCHBr, -OCHI, -OCHF, -OCHCl, -OCHBr, -OCHI, -OCHF, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

140. The method of claim 139, further comprising quantifying the amount of the nucleic acid target based on the measured fluorescent signal.

141. The method of claim 139, wherein the probe comprises a stem-loop portion that is capable of forming a stem-loop structure when the probe is single-stranded.

142. The method of claim 139, wherein Q and R 50 are spaced such that when the probe is single-stranded, R 50 is quenched, but when the probe is incorporated into the double-stranded amplicon, the R 50 is enabled.

143. The method of claim 142, wherein Q and R 50 Both are located at or near the stem-loop portion of the probe.

144. The method of claim 139, wherein the primer pair comprises a primer having a tail.

145. The method of claim 144, wherein the tail forms the 5' end of the primer having the tail.

146. The method of claim 144, wherein the probe is configured to hybridize to the tail or its complement.

147. The method of claim 146, wherein the 3' portion of the probe is configured to hybridize to the tail or its complement.

148. The method of claim 139, wherein the amplification process comprises a denaturation temperature and a plurality of different annealing / extension temperatures that vary throughout the amplification process.

149. A method according to claim 148, wherein a first series of denaturation steps and annealing / extension steps are performed at a first annealing / extension temperature, and wherein a second series of denaturation steps and annealing / extension steps are performed at a different second annealing / extension temperature.

150. The method of claim 149, wherein the first annealing / extension temperature is higher than the second annealing / extension temperature.

151. The method of claim 149, wherein the first series of denaturation steps and annealing / extension steps is cycled more times than the second series of denaturation steps and annealing / extension steps.

152. The method of claim 149, wherein the amplification process further comprises a third series of denaturation steps and annealing / extension steps performed using a third annealing / extension temperature.

153. The method of claim 152, wherein the third annealing / extension temperature is the same as the first annealing / extension temperature.

154. The method of claim 152, wherein the third series of denaturation steps and annealing / extension steps is cycled more times than the first series of denaturation steps and annealing / extension steps.

155. The method of claim 143, wherein the denaturation temperature is the same for each series of denaturation steps and annealing / extension steps.

156. The method of one of claims 144 to 155, wherein the primer pair comprises a tailed primer and a tailless primer, and wherein the tailed primer and the tailless primer are provided at different concentrations.

157. The method of claim 156, wherein the tailless primer is provided at a higher concentration than the tailed primer.

158. The method of claim 157, wherein the tailless primer is provided at a concentration of about 2X to about 30X the concentration of the tailed primer, or at about 5X to about 25X the concentration of the tailed primer, or at a concentration of about 10X to about 20X the concentration of the tailed primer.

159. The method of claim 156, wherein the probe is provided at a concentration that is different from the concentration of the tailed primer and the concentration of the tailless primer.

160. The method of claim 159, wherein the second probe is provided at a concentration lower than the concentration of the tailless primer.

161. The method of claim 159, wherein the probe is provided at a concentration lower than the concentration of the tailless primer.

162. The method of claim 159, wherein the probe is provided at a concentration of about 2X to about 10X the concentration of the tailless primer, or about 3X to about 7.5X the concentration of the tailless primer.

163. A method for detecting nucleic acid in a sample, the method comprising: A reaction mixture is provided, the reaction mixture comprising: a primer pair targeting a nucleic acid target for producing an amplicon, the primer pair comprising a tailed primer and a tailless primer provided at different concentrations, and a non-cleavable probe configured to hybridize to the amplicon, the probe comprising a detectable label configured to generate a fluorescent signal corresponding to the amount of amplicon generated, subjecting the reaction mixture to an amplification process to produce the amplicon, wherein the probe produces fluorescence without being cleaved during the amplification process; and measuring the fluorescent signal from the probe; Wherein the probe has the formula: or a salt thereof, wherein Q is a quencher moiety having the formula: B is a divalent nucleobase; L 1 It is a divalent linker; L 5 is a divalent oligonucleotide linker comprising 4 to 40 nucleotides; L 50 is a bond, -NH-, -O-, -S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; R 50 is a detectable marker; R 30 is - OR 30A ; R 30A is a monovalent oligonucleotide moiety; R 2 is hydrogen or -OR 2A ; R 4 is hydrogen or unsubstituted methyl, or R 2 and R 4 The substituents are linked to form a substituted or unsubstituted heterocycloalkyl group; R 1 and R 10 are independently hydrogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R 6 , R 7 , R 8 and R 9 are independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3R A , -SO2NH2, -NHNH2, -ONH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCBr3, -OCF3, -OCI3, -OCH2Cl, -OCH2Br, -OCH2F, -OCHI, -OCHCl2, -OCHBr2, -OCHF2, -OCHI2, -SF5, -N3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 1 and R 6 Can be linked to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group; R 8 and R 10 capable of being linked to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group; and R 2A and R A and -CHCl, -CHBr, -CHF, -CHI, -CHCl, -CHBr, -CHF, -CHI, -CN, -OH, -NH, -COOH, -CONH, -OCCl, -OCF, -OCBr, -OCI, -OCHCl, -OCHBr, -OCHI, -OCHF, -OCHCl, -OCHBr, -OCHI, -OCHF, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

164. The method of claim 163, wherein the tailless primer is provided at a higher concentration than the tailed primer.

165. The method of claim 164, wherein the tailless primer is provided at a concentration of about 2X to about 30X the concentration of the tailed primer, or at about 5X to about 25X the concentration of the tailed primer, or at a concentration of about 10X to about 20X the concentration of the tailed primer.

166. The method of claim 163, wherein the probe is provided at a concentration that is different from the concentration of the tailed primer and the concentration of the tailless primer.

167. The method of claim 166, wherein the second probe is provided at a concentration lower than the concentration of the tailless primer.

168. The method of claim 166, wherein the probe is provided at a concentration lower than the concentration of the tailless primer.

169. The method of one of claims 166 to 168, wherein the probe is provided at a concentration of about 2X to about 10X the concentration of the tailless primer, or about 3X to about 7.5X the concentration of the tailless primer.

170. A method for detecting the presence or amount of a first target and / or a second target in a reaction mixture, the method comprising: comprising a first probe and a second probe in the reaction mixture, wherein the first probe is capable of specifically interacting with a first target and comprises a first label capable of producing a first detectable signal, and the second probe is capable of specifically interacting with a second target and comprises a second label capable of producing a second detectable signal; Wherein the second probe has the following formula: or a salt thereof, wherein Q is a quencher moiety having the formula: B is a divalent nucleobase; L 1 It is a divalent linker; L 5 is a divalent oligonucleotide linker comprising 4 to 40 nucleotides; L 50 is a bond, -NH-, -O-, -S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; R 50 is the second marker; R 30 is - OR 30A ; R 30A is a monovalent oligonucleotide moiety; R 2 is hydrogen or -OR 2A ; R 4 is hydrogen or unsubstituted methyl, or R 2 and R 4 The substituents are linked to form a substituted or unsubstituted heterocycloalkyl group; R 1 and R 10 are independently hydrogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R 6 , R 7 , R 8 and R 9 are independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3R A , -SO2NH2, -NHNH2, -ONH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCBr3, -OCF3, -OCI3, -OCH2Cl, -OCH2Br, -OCH2F, -OCHI, -OCHCl2, -OCHBr2, -OCHF2, -OCHI2, -SF5, -N3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 1 and R 6 capable of being linked to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group; and R 8 and R 10 capable of being linked to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group; and R 2A and R A are independently hydrogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCHI, -OCH2F, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; allowing the first probe and the second probe to specifically interact with any first target and second target in the reaction mixture, respectively; measuring a first total signal through an optical filter under a first set of conditions, wherein the first total signal includes the first detectable signal and the second detectable signal from the first label and the second label, and wherein under the first set of conditions, the first detectable signal increases due to the specific interaction of the first probe with the first target, but the second detectable signal does not increase due to the specific interaction of the second probe with the second target; measuring a second total signal through the same filter under a second set of conditions, wherein the second total signal includes the first detectable signal and the second detectable signal from the first label and the second label, and wherein under the second set of conditions, the second detectable signal is increased due to the specific interaction of the second probe with the second target; as well as By estimating the first detectable signal and the second detectable signal based on both the first total signal and the second total signal, the presence or amount of the first target and / or the second target is assessed.

171. The method of claim 170, wherein the first marker and the second marker are the same and / or produce substantially the same fluorescence.

172. The method of claim 170, wherein the degree of difference in the second fluorescent signal between the first set of conditions and the second set of conditions is greater than the degree of difference in the first fluorescent signal between the first set of conditions and the second set of conditions.

173. The method of claim 170, wherein the first probe is a cleavable probe.

174. The method of claim 173, wherein the first detectable signal increases when the cleavable probe is cleaved.

175. The method of claim 173, wherein the first probe comprises a fluorophore and a quencher, and wherein the first probe is configured such that fluorescence from the fluorophore is quenched by the quencher until the probe is cleaved.

176. The method of claim 175, wherein the first probe is a TaqMan probe.

177. The method of claim 170, wherein the second probe is a non-cleavable probe.

178. The method of claim 177, wherein when the second probe is single-stranded, L 5 Forming a stem-loop structure.

179. The method of claim 177, wherein R 50 is the fluorophore, and Q and R 50 are spaced apart from each other so that when the second probe is single-stranded, R 50 is quenched, and when the second probe is incorporated into the double-stranded amplicon, the R 50 Not quenched.

180. The method of claim 179, wherein Q and R 50 Both are arranged at or near the stem-loop portion of the second probe.

181. The method of claim 170, wherein the melting temperature (T m ) and the T of the second probe m Within about 8°C, or about 6°C, or about 4°C, or about 2°C of each other.

182. The method of claim 170, wherein the first set of conditions comprises a first measurement temperature at which the first fluorescent signal is measured, and the second set of conditions comprises a different second measurement temperature at which the second fluorescent signal is measured.

183. The method of claim 182, wherein the first measured temperature and the second measured temperature differ by at least about 10°C or more, about 15°C or more, about 20°C or more, about 25°C or more, or about 30°C or more.

184. The method of claim 182, wherein at least one of the first measured temperature or the second measured temperature is a denaturation temperature at which the DNA in the reaction mixture denatures, such as about 90°C or above.

185. The method of one of claims 170 to 184, further comprising thermally cycling the reaction mixture between two target temperatures for a plurality of cycles.

186. A method according to claim 185, wherein the thermal cycle cycles between two target temperatures for at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the cycle.

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